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
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.
T lymphocytes have been implicated in controlling the recruitment of eosinophils into the lung in murine models of allergic asthma. The mechanism by which T cells assist in the recruitment of eosinophils to the lung in these models is not completely understood. We hypothesized that eosinophil-active chemokines might be regulated by antigen (Ag)- induced T cell activation in vivo and thereby mediate T cell-dependent eosinophil recruitment. To test this hypothesis, we examined the effect of an anti-CD3 mAb on Ag-induced pulmonary eosinophilia and correlated this with the expression of three eosinophil-active chemokines: eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and RANTES. We found that Ag-induced pulmonary eosinophilia was associated with the induction of eotaxin and MIP-1 alpha, but not RANTES mRNA. Prechallenge treatment with anti-CD3 mAb inhibited eotaxin, but not MIP-1 alpha and RANTES mRNA induction, and significantly reduced eosinophil accumulation in the lung. In addition, Ag-specific antibody responses and mast cell degranulation after Ag challenge in sensitized mice were not affected by T cell elimination, and were not sufficient to induce the expression of eotaxin and cause pulmonary eosinophilia. These findings suggest that eotaxin is one of the molecular links between Ag- specific T cell activation and the recruitment of eosinophils into the airways.
Heparan sulfate (HS) proteoglycans (HSPGs) participate in several aspects of inflammation because of their ability to bind to growth factors, chemokines, interleukins and extracellular matrix proteins as well as promote inflammatory cell trafficking and migration. We investigated whether HSPGs play a role in the development of airway remodeling during chronic allergic asthma using mice deficient in endothelial- and leukocyte-expressed N-deacetylase/N-sulfotransferase-1 (Ndst1), an enzyme involved in modification reactions during HS biosynthesis. Ndst1-deficient and wild-type (WT) mice exposed to repetitive allergen (ovalbumin [OVA]) challenge were evaluated for the development of airway remodeling. Chronic OVA-challenged WT mice exhibited increased HS expression in the lungs along with airway eosinophilia, mucus hypersecretion, peribronchial fibrosis, increased airway epithelial thickness and smooth muscle mass. In OVA-challenged Ndst1-deficient mice, lung eosinophil and macrophage infiltration as well as airway mucus accumulation, peribronchial fibrosis and airway epithelial thickness were significantly lower than in allergen-challenged WT mice along with a trend toward decreased airway smooth muscle mass. Leukocyte and endothelial Ndst 1 deficiency also resulted in significantly decreased expression of IL-13 as well as remodeling-associated mediators such as VEGF, FGF-2 and TGF-β1 in the lung tissue. At a cellular level, exposure to eotaxin-1 failed to induce TGF-β1 expression by Ndst1-deficient eosinophils relative to WT eosinophils. These studies suggest that leukocyte and endothelial Ndst1-modified HS contribute to the development of allergen-induced airway remodeling by promoting recruitment of inflammatory cells as well as regulating expression of pro-remodeling factors such as IL-13, VEGF, TGF-β1 and FGF-2 in the lung.
allergen-induced airway remodeling; FGF-2;
heparan sulfates; N-Deacetylase/N-Sulfotransferase-1; TGF-β
The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness
(BHR) that characterize asthma is achieved by the regulated accumulation and activation of
different leukocyte subsets in the lung. The development and maintenance of these processes
correlate with the coordinated production of chemokines. Here, we have assessed the role that
different chemokines play in lung allergic inflammation and BHR by blocking their activities
in vivo. Our results show that blockage of each one of these chemokines reduces both lung
leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the
eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES
(regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor
antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1α, reduces only slightly lung eosinophilia and BHR.
Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates
with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators.
These results suggest that different chemokines activate different cellular and molecular pathways
that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their
individual blockage results in intervention at different levels of these processes.
chemokines; allergic inflammation; bronchial hyperresponsiveness; eosinophilia; leukocytes
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
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
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.
The effect of targeted inactivation of the gene encoding N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in the biosynthesis of heparan sulfate (HS) chains, on the inflammatory response associated with allergic inflammation in a murine model of OVA-induced acute airway inflammation was investigated. OVA-exposed Ndst1f/fTekCre+ (mutant) mice deficient in endothelial and leukocyte Ndst1 demonstrated significantly decreased allergen-induced airway hyperresponsiveness and inflammation characterized by a significant reduction in airway recruitment of inflammatory cells (eosinophils, macrophages, neutrophils, and lymphocytes), diminished IL-5, IL-2, TGF-β1, and eotaxin levels, as well as decreased expression of TGF-β1 and the angiogenic protein FIZZ1 (found in inflammatory zone 1) in lung tissue compared with OVA-exposed Ndst1f/fTekCre− wild-type littermates. Furthermore, murine eosinophils demonstrated significantly decreased rolling on lung endothelial cells (ECs) from mutant mice compared with wild-type ECs under conditions of flow in vitro. Treatment of wild-type ECs, but not eosinophils, with anti-HS Abs significantly inhibited eosinophil rolling, mimicking that observed with Ndst1-deficient ECs. In vivo, trafficking of circulating leukocytes in lung microvessels of allergen-challenged Ndst1-deficient mice was significantly lower than that observed in corresponding WT littermates. Endothelial-expressed HS plays an important role in allergic airway inflammation through the regulation of recruitment of inflammatory cells to the airways by mediating interaction of leukocytes with the vascular endothelium. Furthermore, HS may also participate by sequestering and modulating the activity of allergic asthma-relevant mediators such as IL-5, IL-2, and TGF-β1.
Crataegus pinnatifida (Chinese hawthorn) has long been used as a herbal medicine in Asia and Europe. It has been used for the treatment of various cardiovascular diseases such as myocardial weakness, tachycardia, hypertension and arteriosclerosis. In this study, we investigated the anti-inflammatory effects of Crataegus pinnatifida ethanolic extracts (CPEE) on Th2-type cytokines, eosinophil infiltration, expression of matrix metalloproteinase (MMP)-9, and other factors, using an ovalbumin (OVA)-induced murine asthma model.
Airways of OVA-sensitized mice exposed to OVA challenge developed eosinophilia, mucus hypersecretion and increased cytokine levels. CPEE was applied 1 h prior to OVA challenge. Mice were administered CPEE orally at doses of 100 and 200 mg/kg once daily on days 18–23. Bronchoalveolar lavage fluid (BALF) was collected 48 h after the final OVA challenge. Levels of interleukin (IL)-4 and IL-5 in BALF were measured using enzyme-linked immunosorbent (ELISA) assays. Lung tissue sections 4 µm in thickness were stained with Mayer’s hematoxylin and eosin for assessment of cell infiltration and mucus production with PAS staining, in conjunction with ELISA, and Western blot analyses for the expression of MMP-9, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 protein expression. CPEE significantly decreased the Th2 cytokines including IL-4 and IL-5 levels, reduced the number of inflammatory cells in BALF and airway hyperresponsiveness, suppressed the infiltration of eosinophil-rich inflammatory cells and mucus hypersecretion and reduced the expression of ICAM-1, VCAM-1 and MMP-9 and the activity of MMP-9 in lung tissue of OVA-challenged mice.
These results showed that CPEE can protect against allergic airway inflammation and can act as an MMP-9 modulator to induce a reduction in ICAM-1 and VCAM-1 expression. In conclusion, we strongly suggest the feasibility of CPEE as a therapeutic drug for allergic asthma.
Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-deficient C57BL/6 ΔdblGATA mice (eosinophil-null mice via the ΔDblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway inflammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-deficient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infiltration and airway inflammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.
The chemokines are a large group of chemotactic cytokines that regulate leukocyte trafficking and have recently been shown to inhibit human immunodeficiency virus entry into cells. Eotaxin is a C–C chemokine implicated in the recruitment of eosinophils in a variety of inflammatory disorders and, unlike all other eosinophil chemoattractants, is eosinophil specific. However, given the large number of chemoattractants that have activities on eosinophils, it is unclear whether eotaxin has an important role in vivo. Furthermore, it remains unclear why there is constitutive expression of eotaxin in healthy states in the absence of eosinophilic inflammation. To begin to determine the significance of eotaxin at baseline and during eosinophil-mediated disease processes, we have used targeted gene disruption to generate mice that are deficient in eotaxin. Such mice demonstrate that eotaxin enhances the magnitude of the early (but not late) eosinophil recruitment after antigen challenge in models of asthma and stromal keratitis. Surprisingly, a role for eotaxin in regulating the constitutive number of eosinophils in the peripheral circulation is also demonstrated. These results indicate a contributory role for eotaxin in the generation of peripheral blood and antigen-induced tissue eosinophilia.
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
Tissue eosinophil infiltration, which is a hallmark of allergic and helminthic diseases, is mainly coordinated by T lymphocytes, via the production of eosinophilotactic chemokines. Among T lymphocyte subsets, lymphocytes expressing γδ T cell receptor have been determined as a key factor for eosinophil accumulation via direct and indirect mechanisms. This knowledge is strongly supported by the fact that, in different experimental models of eosinophilic airway inflammation and helminth-induced Th2 lung inflammation, an evident tissue accumulation of γδ T lymphocytes is observed. In addition, the depletion of γδ T lymphocytes is correlated with the impairment of eosinophil accumulation in inflamed tissue. γδ T lymphocytes are non-conventional T lymphocytes, which comprise a minor T lymphocyte subset, mainly distributed in the tissue, and present crucial roles in innate and acquired immune responses. γδ T lymphocytes recognize several danger- and pathogen-associated molecular pattern molecules and stress antigens in a MHC-independent fashion and can provide rapid tissue-specific responses, via the production of a wide range of chemical mediators capable to modulate other cell populations. These mediators include chemoattractant cytokines and chemokines that attract eosinophils into the tissue by either direct recognition (such as IL-5, CCL11/eotaxin), or indirect mechanisms via the modulation of αβ T lymphocytes and macrophages (through the production of interferon-γ, IL-4, and CCL2/Monocyte chemoattractant protein-1, MCP-1, for example). The present review presents an overview of how γδ T lymphocytes coordinate eosinophil accumulation in allergy, by focusing on their role in airway inflammation and by discussing the involvement of cytokines and chemokines in this phenomenon.
integrin α4β1; selectins; MCP-1; IL-17; leukotriene B4
Asthma is a chronic inflammatory disease induced by Type 2 helper T cells and eosinophils. Vascular cell adhesion molecule-1 (VCAM-1) has been implicated in recruiting eosinophils and lymphocytes to pathological sites in asthma as a regulatory receptor. Accordingly, monoclonal antibody (mAb) against VCAM-1 may attenuate allergic inflammation and pathophysiological features of asthma. We attempted to evaluate whether a recently developed human anti-VCAM-1 mAb can inhibit the pathophysiological features of asthma in a murine asthma model induced by ovalbumin (OVA). Leucocyte adhesion inhibition assay was performed to evaluate the in vitro blocking activity of human anti-VCAM-1 mAb. OVA-sensitized BALB/c mice were treated with human anti-VCAM-1 mAb or isotype control Ab before intranasal OVA challenge. We evaluated airway hyperresponsiveness (AHR) and bronchoalveolar lavage fluid analysis, measured inflammatory cytokines and examined histopathological features. The human anti-VCAM-1 mAb bound to human and mouse VCAM-1 molecules and inhibited adhesion of human leucocytes in vitro. AHR and inflammatory cell counts in bronchoalveolar lavage fluid were reduced in mice treated with human anti-VCAM-1 mAb as compared with a control Ab. The levels of interleukin (IL)-5 and IL-13, as well as transforming growth factor-β, in lung tissue were decreased in treated mice. Human anti-VCAM-1 mAb reduced goblet cell hyperplasia and peribronchial fibrosis. In vivo VCAM-1 expression decreased in the treated group. In conclusion, human anti-VCAM-1 mAb attenuated allergic inflammation and the pathophysiological features of asthma in OVA-induced murine asthma model. The results suggested that human anti-VCAM-1 mAb could potentially be used as an additional anti-asthma therapeutic medicine.
VCAM-1; monoclonal antibody; allergic inflammation; asthma; cell adhesion molecule; anti-inflammatory effect
Airway eosinophilia and Th2 lymphocytes-recruitment to the lung are one of the main pathological features of asthma. It is clear now that the axis chemokine/chemokine receptors have a role in controlling leukocyte recruitment and development of the inflammatory process observed in asthma. Although it has been reported that CCR9 receptor is expressed in asthmatic patients, it is not known whether CCR9 may have a regulatory role of the development of this disease. Our aim was to analyze the expression of CCR9 in a murine model of allergic airway inflammation (WT) and compared to CCR9 deficient (KO) mice.
Four groups of 6 to 8 weeks female CCR9-deficient mice were sensitized by intraperitoneal injections of 10 micrograms of ovalbumin (OVA) in alum (ALOH3) diluted in PBS, on days 1 and 8 of the established sensitization protocol. Aerosolised OVA was administered (1% in PBS) on days 15, 20 and 34. 24 hours after last OVA exposure, mice were sacrificed and bronchoalveolar lavage (BAL) fluid and cells were obtained. Total and differential cell numbers were obtained and characterized cell subpopulations by FACS analysis. Cytokine/chemokine levels were quantified by ELISA and qRT-PCR respectively.
Total cell numbers in BAL were no significantly different between WT and KO mice. Interestingly, reduction in the numbers of eosinophils was observed in CCR9 KO mice compared to WT mice. Histological analysis of lung tissue demonstrated a reduction in the granulocytic population (eosinophils) in CCR9 KO mice. Analysis of cell subpopulations by FACS demonstrated that CD4+ lymphocytes were significantly reduced but CD8+ and CD19+ lymphocytes numbers were not different between WT and CCR9-deficient mice. A population of CCR9+ Gr1+ was altered in KO mice and it correlated with cytological analysis. Furthermore, histological analysis demonstrated alteration in mucus production in allergic airway in CCR9 deficient mice, accompanied with a no-significant reduction of OVA-specific anti-IgE antibodies in serum at the time of analysis.
Altogether, these results suggest that CCR9 may be involved in recruitment of granulocytic cell subpopulation into the allergic airways and have an impact in the regulation of the chronic inflammatory process.
Overactivation of nuclear factor κB (NF-κB) orchestrates airway eosinophilia, but does not dampen airway hyperresponsiveness in asthma. NF-κB repression by arsenic trioxide (As2O3) contributes to apoptosis of eosinophils (EOS) in airways. Here we provide evidence that As2O3 abrogates allergen (OVA)-induced airway eosinophilia by modulating the expression of IκBα, an NF-κB inhibitory protein, and decreases the airway hyperresponsiveness.
Using a murine model of asthma, the airway hyperresponsiveness was conducted by barometric whole-body plethysmography. Airway eosinophilia, OVA-specific IgE in serum, and chemokine eotaxin and RANTES (regulated upon activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid were measured by lung histology, Diff-Quick staining, and ELISA. Chemokine-induced EOS chemotactic activity was evaluated using EOS chemotaxis assay. Electrophoretic mobility shift assay and Western blot analysis were performed to assess pulmonary NF-κB activation and IκBα expression, respectively.
As2O3 attenuated the allergen-induced serum IgE, chemokine expression of eotaxin and RANTES, and the EOS recruitment in bronchoalveolar lavage fluid, which is associated with an increased IκBα expression as well as a decreased NF-κB activation. Also, As2O3 suppressed the chemotaxis of EOS dose-dependently in vitro. Additionally, As2O3 significantly ameliorated the allergen-driven airway hyperresponsiveness, the cardinal feature underlying asthma.
These findings demonstrate an essential role of NF-κB in airway eosinophilia, and illustrate a potential dissociation between airway inflammation and hyperresponsiveness. As2O3 likely exerts its broad anti-inflammatory effects by suppression of NF-κB activation through augmentation of IκBα expression in asthma.
Previous work from our laboratory demonstrated that IL-4Rα expression on a myeloid cell type was responsible for enhancement of Th2-driven eosinophilic inflammation in a mouse model of allergic lung inflammation. Subsequently, we have shown that IL-4 signaling through type I IL-4 receptors on monocytes/macrophages strongly induced activation of the IRS-2 pathway and a subset of genes characteristic of alternatively activated macrophages. The direct effect(s) of IL-4 and IL-13 on mouse eosinophils are not clear. The goal of this study was determine the effect of IL-4 and IL-13 on mouse eosinophil function.
Standard Transwell chemotaxis assay was used to assay migration of mouse eosinophils and signal transduction was assessed by Western blotting.
Here we determined that (i) mouse eosinophils express both type I and type II IL-4 receptors, (ii) in contrast to human eosinophils, mouse eosinophils do not chemotax to IL-4 or IL-13 although (iii) pre-treatment with IL-4 but not IL-13 enhanced migration to eotaxin-1. This IL-4-mediated enhancement was dependent on type I IL-4 receptor expression: γC-deficient eosinophils did not show enhancement of migratory capacity when pre-treated with IL-4. In addition, mouse eosinophils responded to IL-4 with the robust tyrosine phosphorylation of STAT6 and IRS-2, while IL-13-induced responses were considerably weaker.
The presence of IL-4 in combination with eotaxin-1 in the allergic inflammatory milieu could potentiate infiltration of eosinophils into the lungs. Therapies that block IL-4 and chemokine receptors on eosinophils might be more effective clinically in reducing eosinophilic lung inflammation.
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.
IL-5 is induced locally in the lung and systemically in the circulation during allergic airways eosinophilic inflammation both in humans and experimental animals. However, the precise role of local and systemic IL-5 in the development of allergic airways eosinophilia remains to be elucidated. In our current study, we demonstrate that compared with their IL-5(+/+) counterparts, IL-5(-/-) mice lacked an IL-5 response both in the lung and peripheral blood, yet they released similar amounts of IL-4, eotaxin, and MIP-1alpha in the lung after ovalbumin (OVA) sensitization and challenge. At cellular levels, these mice failed to develop peripheral blood and airways eosinophilia while the responses of lymphocytes, neutrophils, and macrophages remained similar to those in IL-5(+/+) mice. To dissect the relative role of local and systemic IL-5 in this model, we constructed a gene transfer vector expressing murine IL-5. Intramuscular IL-5 gene transfer to OVA-sensitized IL-5(-/-) mice led to raised levels of IL-5 compartmentalized to the circulation and completely reconstituted airways eosinophilia upon OVA challenge, which was associated with reconstitution of eosinophilia in the bone marrow and peripheral blood. Significant airways eosinophilia was observed for at least 7 d in these mice. In contrast, intranasal IL-5 gene transfer, when rendered to give rise to a significant but compartmentalized level of transgene protein IL-5 in the lung, was unable to reconstitute airways eosinophilia in OVA-sensitized IL-5(-/-) mice upon OVA-challenge, which was associated with a lack of eosinophilic responses in bone marrow and peripheral blood. Our findings thus provide unequivocal evidence that circulating but not local lung IL-5 is critically required for the development of allergic airways eosinophilia. These findings also provide the rationale for developing strategies to target circulating IL-5 and/or its receptors in bone marrow to effectively control asthmatic airways eosinophilia.
Eosinophils are the predominant inflammatory cells recruited to allergic airways. Here we demonstrate that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared to WT eosinophils, SWAP-70 deficient (Swap-70−/−) eosinophils revealed altered adhesive interactions within inflamed post capillary venules under conditions of blood flow by intravital microscopy exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70−/− eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70−/− eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70−/− eosinophils was significantly decreased correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization and altered cytoskeletal rearrangement. In vivo, compared to WT mice, recruitment of eosinophils to the lungs of allergen-challenged Swap-70−/− mice was significantly reduced along with considerable attenuation of airway inflammation indicated by diminished IL-5, IL-13 and TNFα levels, reduced mucus secretion and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.
It is widely assumed that apoptosis of eosinophils is a central component of resolution of allergic airway disease. However, this has not been demonstrated in human allergic airways in vivo. Based on animal in vivo observations we hypothesised that steroid-induced resolution of human airway eosinophilic inflammation involves inhibition of CCL5 (RANTES), a CC-chemokine regulating eosinophil and lymphocyte traffic, and elimination of eosinophils without evident occurrence of apoptotic eosinophils in the diseased tissue.
To determine mucosal eosinophilia, apoptotic eosinophils, general cell apoptosis and cell proliferation, and expression of CCL5 and CCL11 (eotaxin) in human allergic airway tissues in vivo at resolution of established symptomatic eosinophilic inflammation.
Twenty-one patients with intermittent (birch and/or grass) allergic rhinitis received daily nasal allergen challenges for two seven days' periods separated by more than two weeks washout. Five days into these "artificial pollen seasons", nasal treatment with budesonide was instituted and continued for six days in a double blinded, randomized, placebo-controlled, and crossover design. This report is a parallel group comparison of nasal biopsy histochemistry data obtained on the final day of the second treatment period.
Treatments were instituted when clinical rhinitis symptoms had been established. Compared to placebo, budesonide reduced tissue eosinophilia, and subepithelial more than epithelial eosinophilia. Steroid treatment also attenuated tissue expression of CCL5, but CCL11 was not reduced. General tissue cell apoptosis and epithelial cell proliferation were reduced by budesonide. However, apoptotic eosinophils were not detected in any biopsies, irrespective of treatment.
Inhibition of CCL5-dependent recruitment of cells to diseased airway tissue, and reduced cell proliferation, reduced general cell apoptosis, but not increased eosinophil apoptosis, are involved in early phase steroid-induced resolution of human allergic rhinitis.
Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils.
Eosinophils were purified using a percoll gradient followed by immunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry.
At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils.
Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion.
This study was undertaken to investigate the possible contribution of the blockade of eotaxin generation to the anti-eosinophilotactic effect of phosphodiesterase (PDE) type 4 inhibitors. In some experiments, the putative synergistic interaction between PDE type 4 inhibitors and the β2-agonist salbutamol was also assessed.Sensitized guinea-pigs aerosolized with antigen (5% ovalbumin, OVA) responded with a significant increase in eotaxin and eosinophil levels in the bronchoalveolar lavage fluid (BALF) at 6 h. Eosinophil recruitment was inhibited by both PDE type 4 inhibitors rolipram (5 mg kg−1, i.p.) and RP 73401 (5 mg kg−1, i.p.) treatments. In contrast, only rolipram inhibited eotaxin production.Sensitized rats intrapleurally challenged (i.pl.) with antigen (OVA, 12 μg cavity−1) showed a marked eosinophil infiltration at 24 h, preceded by eotaxin generation at 6 h. Intravenous administration of a rabbit anti-mouse eotaxin antibody (0.5 mg kg−1) significantly reduced allergen-evoked eosinophilia in this model.Local pretreatment with rolipram (40 μg cavity−1) or RP 73401 (40 μg cavity−1) 1 h before challenge reduced eosinophil accumulation evaluated in the rat pleural effluent, but only the former was active against eotaxin generation. The inhibitors of PDE type 3 (SK&F 94836) and type 5 (zaprinast) failed to alter allergen-evoked eosinophil recruitment in rats.Local injection of β2-agonist salbutamol (20 μg cavity−1) inhibited both eosinophil accumulation and eotaxin production following pleurisy. The former was better inhibited when salbutamol and rolipram were administered in combination.Treatment with rolipram and RP 73401 dose-dependently inhibited eosinophil adhesion and migration in vitro. These effects were clearly potentiated by salbutamol at concentrations that had no effect alone.Our findings indicate that although rolipram and RP 73401 are equally effective in inhibiting allergen-induced eosinophil infiltration only the former prevents eotaxin formation, indicating that PDE 4 inhibitors impair eosinophil accumulation by mechanisms independent of eotaxin production blockade.
Eosinophil migration and adhesion; eotaxin; allergic inflammation; PDE 4 inhibitors