Airway epithelial cells promote transmigration of eosinophils in a new three-dimensional chemotaxis model

BACKGROUND: Prominent infiltration of eosinophils in airway mucosa is the pathognomonic sign of asthma. The role of airway epithelial cells in eosinophil infiltration, however, has not been fully elucidated. OBJECTIVE: The aim of this study is to develop a new in vitro transmigration system composed of airway epithelial cells and extracellular matrix, and to investigate the role of airway epithelial cells in eosinophil infiltration. METHODS: A layer of type I collagen gel was formed in Netwell, and BEAS-2B bronchial epithelial cells were cultured on the gel. Then the wells covered with epithelial monolayer were filled with medium, inverted, and new upper chambers were constructed on the gel side by applying a ring cap. After further incubation with or without exogenous cytokines for 48 h, eosinophils or neutrophils were loaded in upper chambers (the gel side) and cells transmigrated to lower chambers (the epithelial cell side) were counted. Immunohistochemical analyses were also performed. RESULTS: While a simple collagen gel hardly promoted eosinophil migration even in the presence of eotaxin or RANTES, significant numbers of eosinophils migrated to lower chambers in the presence of the epithelial cells. Replacement of medium in the lower chamber (the epithelial cell side) with fresh medium, addition of exogenous eotaxin or RANTES in the upper chamber (the gel side), or pre-treatment of eosinophils with anti-CCR3 all inhibited transmigration. We found that the epithelial cells produced and deposited extracellular matrix proteins such as type IV collagen onto the type I collagen gel. Separately, we found that type IV collagen itself was capable of enhancing eotaxin-induced eosinophil migration in a standard chemotaxis assay. Neutrophils also efficiently migrated in the present transmigration system. Pre-treatment of epithelial cells with TNF-alpha and IL-4 enhanced eosinophil transmigration, while that of neutrophils was enhanced by TNF-alpha but suppressed by IL-4. CONCLUSION: By utilizing a new in vitro transmigration system mimicking the airway mucosa, we have demonstrated that airway epithelial cells play an essential role in transmigration of eosinophils and that multiple factors such as chemokines, extracellular matrix proteins and exogenous inflammatory cytokines are involved in efficient transmigration.     

Immune effector functions of eosinophils in allergic airway inflammation

Eosinophils have the capacity to exert immune effector actions on the airways of asthmatic individuals by virtue of their ability to elaborate cytokines, chemokines and growth factors. The present review discusses cutting edge research on eosinophil cytokine release and its potential role in allergic airway inflammation.     

Degranulation status of airway tissue eosinophils in mouse models of allergic airway inflammation

Eosinophil degranulation is a characteristic feature of asthma and allergic rhinitis. However, degranulated eosinophils have not been convincingly demonstrated in the common mouse models of these airway diseases. This study uses eosinophil peroxidase (EPO) histochemistry and transmission electron microscopy (TEM) analysis to assess eosinophil degranulation in the airways of ovalbumin (OVA)-sensitized and challenged BALB/c and C57BL/6 mice. Using TEM we also examined mouse and human blood eosinophils after in vitro incubation with formyl-Met-Leu-Phe (fMLP) or phorbol myristate acetate (PMA). Although OVA exposure induced significant nasal and lung eosinophilia, we did not observe any of the known cellular processes by which eosinophils release their granule products, i.e., eosinophil cytolysis, piecemeal degranulation, and exocytosis. The occurrence of other allergen-induced degranulation events was ruled out because no difference in granule morphology was observed between lung-tissue eosinophils and blood or bone-marrow eosinophils from control animals. Accordingly, there was no detectable extracellular EPO in lung tissues of allergic mice. Similarly, mouse blood eosinophils remained nondegranulated in vitro in the presence of fMLP and PMA, whereas the same treatment of human eosinophils resulted in extensive degranulation. This investigation indicates that OVA-induced airway inflammation in the present mouse strains does not involve significant eosinophil degranulation. It is speculated that this dissimilarity from the human disease may be due to a fundamental difference in the regulation of mouse and human eosinophils.     

Diesel exhaust particles stimulate human airway epithelial cells to produce cytokines relevant to airway inflammation in vitro

Background: Epidemiologic and experimental studies suggest that air pollution such as diesel exhaust particles (DEPs), one of the important air pollutants, may play a role in the increasing prevalence of allergic airway diseases. Objective: We studied the effect of suspended particulate matter (SPM) and its main component, DEPs, on the production of IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by human airway epithelial cells in vitro. Methods: SPM obtained from high-volume air samplers and DEPs were added to cultured human nasal polyp–derived upper airway, normal bronchial, and transformed bronchial epithelial cells. Production of GM-CSF and IL-8 by airway epithelial cells was evaluated. Results: Nontoxic doses of DEPs showed a significant stimulatory effect on IL-8 and GM-CSF production by these three kinds of epithelial cells in a dose- and time-dependent fashion. SPM had a stimulatory effect on GM-CSF, but not IL-8, production. These effects were abrogated by treatment with a protein synthesis inhibitor, cycloheximide, suggesting that the process required a de novo protein synthesis. On the double-chamber plates, airway epithelial cells responded to DEPs only when they were stimulated from the apical sides, which can be a model for in vivo environments. Neither charcoal nor graphite showed such stimulatory effects, indicating that the activity of DEPs did not derive from their particulate nature. Benzo(a)pyrene, one of the main aromatic hydrocarbons contained in DEPs, showed a stimulatory effect on the release of the cytokines, and this organic substance might have a causative effect on of the potency of DEPs. Conclusion: We conclude that SPM and DEPs, its main component, might be important air pollutants in the activation of airway epithelial cells for the release of cytokines relevant to allergic airway inflammation. (J Allergy Clin Immunol 1998;101:778-785.)     

Mast cells and eosinophils: the two key effector cells in allergic inflammation

The allergic inflammatory response is composed of two main phases—the early and the late. The early phase initiates when an allergen activates the tissue resident mast cell, triggering the release of a variety of granule-stored and newly formed mediators. As the inflammatory response progresses, blood borne inflammatory cells—in particular, eosinophils—are recruited into the inflamed tissue. Eosinophil activation and consequent release and production of several pro-inflammatory mediators results in the late phase reaction. A chronic allergic inflammation always features prominent tissue eosinophilia. In this review, we will discuss the possible channels of communication, both soluble and physical, between mast cells and eosinophils that can occur in the late and chronic stages of allergy. Such interactions, that we have termed “the allergic effector unit”, may modulate the severity and/or duration of the allergic inflammatory reaction.     

Increased surfactant protein D in rat airway goblet and Clara cells during ovalbumin-induced allergic airway inflammation

BACKGROUND: Structural remodelling of airways in asthma that follows inflammation may be affected by surfactant protein D (SP-D)-mediated effects on the immune response. OBJECTIVE: To determine potential sites of SP-D interaction with the pulmonary immune response, we examined the distribution of immunoreactive SP-D in an experimental model of allergen-induced airway inflammation using immunohistochemistry, biochemical methods and in situ hybridization. METHODS: The experimental model used subcutaneous injection of ovalbumin in adult rats, which induced an airway response to inhaled nebulized ovalbumin. Three groups of rats (ovalbumin, ovalbumin + dexamethasone and saline) were challenged thrice weekly for 3 weeks. A fourth group of seven rats (naive) were taken from the same delivery of rats as the other groups. Lungs were then lavaged to determine total cell count, eosinophil count, ovalbumin-specific IgE by enzyme-linked immunosorbent assay and SP-D by immunoblot. Tissue samples were fixed and embedded, and sections were studied for the infiltration of eosinophils and for expression of SP-D protein by histochemistry and mRNA by in situ hybridization. RESULTS: Ovalbumin induced perivascular and peribronchiolar eosinophilia which could be prevented by dexamethasone treatment. In addition, the ovalbumin-specific IgE levels in serum and bronchoalveolar lavage fluid of ovalbumin-challenged animals were enhanced. Increased amount of SP-D in lavage and tissue, particularly in type II pneumocytes, in Clara cells and, surprisingly, in hyperplastic goblet cells of inflamed lungs was found. SP-D mRNA was detected in goblet cells as well as in type II pneumocytes and Clara cells. Dexamethasone treatment did not affect level of SP-D immunoreactivity. CONCLUSION: SP-D accumulation is increased in this model of allergen-induced eosinophilia, both in upper and lower airways. The increase is unaffected by dexamethasone.     

Deficiency of regulatory B cells increases allergic airway inflammation

Objective: To investigate the effect of the X-linked immunodeficiency (Xid) B cell defect on the response to the cockroach allergen in mice. Methods: Two cockroach allergen immunization and challenge protocols were employed to sensitize CBA/J wild-type and CBA/CaHN-btk(-/-)xid/J (Xid) mice. Blood and tissue samples were collected 24 and 48 hrs after the last intratracheal antigen challenge and were analyzed for several parameters of allergic inflammation. Results: Nearly equivalent amounts of serum IgE were detected in Xid and CBA/J mice after short-term antigen challenge despite the B cell deficiency in Xid mice. A decreased concentration of IgE was detected in CBA/J mice after repeated allergen challenges but not in the Xid mice. Correlating with the discrepancy in serum IgE levels, higher levels of IL-13, IL-5, IL-10 and CCL5 were measured in whole lung homogenates from allergen-challenged Xid mice compared to CBA/J mice. In addition, draining lymph node cells from Xid mice expressed elevated levels of IL-4, IL-5, IL-10 and IFNγ mRNA compared to cells from CBA/J mice after in vitro culture with cockroach antigen. An increase in lung inflammation, interstitial eosinophilia and mucus production was also observed in allergen-challenged Xid mice. CD95L expression increased on B-1a cells following allergen challenge, which was accompanied by an increase in lung CD4⁺ Th cell apoptosis in wild-type CBA/J mice. In contrast, Xid mice did not have an increase in CD4⁺ T cell apoptosis following allergen challenge. Conclusions: These data suggest a regulatory role for B-1a cells in reducing cytokine production, pulmonary inflammation, and CD4⁺ T cell survival during cockroach allergen-induced airway inflammation. Received 10 June 2005; returned for revision 13 September 2005; accepted by M. Parnham 14 September 2005 Funding source: U. S. Government NIH Grant# AI36302     

Interleukin-17 as a recruitment and survival factor for airway macrophages in allergic airway inflammation

Recent data indicate that the proinflammatory cytokine, interleukin (IL)-17, stimulates certain effector functions of human macrophages. We evaluated whether IL-17 mediates allergen-induced accumulation of airway macrophages and, if so, whether such an effect relates to the control of macrophage recruitment and survival. BALB/c mice were sensitized and challenged with ovalbumin. Three hours before challenge an anti-mouse IL-17 mAb (a-IL-17) was administered. Sampling was conducted 24 h after the allergen challenge. In vitro chemotaxis assay for blood monocytes and culture of airway macrophages, immunocytochemistry for Fas-antigen, and matrix metalloproteinase-9 (MMP-9) were used to determine the effect of IL-17 on the recruitment, survival, and activity of airway macrophages. A-IL-17 reduced the number of airway neutrophils and macrophages after allergen challenge. In vitro, recombinant IL-17 induced migration of blood monocytes and prolonged survival of airway macrophages. A-IL-17 also increased the expression of Fas-antigen in airway macrophages in vivo. Finally, the expression of MMP-9 by airway neutrophils and macrophages in vivo was downregulated by a-IL-17. This study indicates that endogenous IL-17 mediates the accumulation of macrophages during allergen-induced airway inflammation. IL-17 exerts its effects by acting directly on airway macrophages by promoting their recruitment and survival. Furthermore, IL-17 is involved in controlling the proteolytic activity of macrophages and neutrophils in allergen-induced airway inflammation.     

An inactivated Pseudomonas aeruginosa medicament inhibits airway allergic inflammation and improves epithelial functions

The features of asthma are airway hyperresponsiveness (AHR), excess production of Th2 cytokines, and eosinophil accumulation in the lungs. To investigate the antiasthmatic potential of an inactivated Pseudomonas aeruginosa medicament (PPA), as well as the underlying mechanism involved, we studied the effects of PPA on airway and epithelial functions. Airway resistance, cell enumeration, and IL-4, IFN-γ, and IL-17 secretion in bronchoalveolar lavage fluid were assayed on an OVA-sensitized AHR animal model. Flow cytometry was used to observe the effects of PPA on cell proliferation, real-time PCR was used to test the expressions of toll like receptor 4 and 5, and Th17 signal molecules Act1, NF-kB negative regulator A20, and western blot were used to detect NF-kB expression on cultured human bronchial epithelial cells (BECs). PPA-treated animals had suppressed airway resistance, eosinophil and lymphocytes infiltration, and IL-4 and IL-17 secretion. PPA can stimulate toll-like receptor-4 and 5 expressions, promote cell proliferation in normal and OVA-treated BECs, significantly decrease Act1 and NF-kB, and increase A20 expression in BECs treated by OVA. Our data suggest the therapeutic mechanism by which PPA effectively treats allergic inflammation on reductions of airway responsiveness, eosinophil infiltration, IL-4 and IL-17 secretion, and improvements of epithelial functions.     

Apoptosis of eosinophils and lymphocytes in allergic inflammation.

A characteristic feature of apoptosis is that the cellular contents that are biologically active are always surrounded by the cell membrane throughout the entire process. Thus the apoptotic cells are eliminated calmly and quickly without evoking inflammation. In allergic inflammation activated eosinophils and lymphocytes have been known to accumulate at the site of inflammation at least in part because of their prolonged survival. Corticosteroids are the most potent anti-inflammatory agent used for treating asthma. They inhibit the prolonged survival of eosinophils and lymphocytes directly by inducing apoptosis and indirectly by suppressing the release of cytokines supporting their survival. Theophylline, a classical bronchodilator, has been reported to have anti-inflammatory effects. Surprisingly, theophylline inhibited the prolonged survival of eosinophils in the presence of IL-5 in vitro by means of induction of apoptosis. Lymphocytes also undergo apoptosis in the presence of theophylline. One of the mechanisms for theophylline to induce apoptosis in eosinophils and lymphocytes is to elevate intracellular cyclic adenosine monophosphate because phosphodiesterase inhibitors and adenylate cyclase activators, which increase intracellular cyclic adenosine monophosphate, can cause apoptosis in those cells. Induction of apoptosis is beneficial in allergic inflammation, and the use of corticosteroids and theophylline in combination may be appropriate to induce apoptosis in eosinophils and lymphocytes.     

The origins of basophils and eosinophils in allergic inflammation

The bone marrow actively participates in the production of IgE-positive inflammatory cells (eosinophils, basophils, and mast cells), which are typically recruited to tissues in atopic individuals. Understanding the signaling between the tissue and the bone marrow at the molecular level may well open up new avenues of therapy for allergic inflammation. (J Allergy Clin Immunol 1998;102:S74-6.)     

Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, alphavbeta3, and alphavbeta5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1beta and TNF-alpha were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation.     

Airway mast cells and eosinophils correlate with clinical severity and airway hyperresponsiveness in corticosteroid-treated asthma

BACKGROUND: The relationship between airway inflammation and asthma severity in corticosteroid-treated asthma is unclear. OBJECTIVES: Our purpose was to characterize the inflammatory cell profile of the airway lumen and epithelium in corticosteroid-treated asthma and to relate these findings to clinical and physiologic markers of asthma severity. METHODS: Adults (n = 20) with asthma received standardized high-dose inhaled corticosteroid therapy with beclomethasone 2000 microgram per day for 8 weeks. Airway responsiveness to methacholine and hypertonic (4.5%) saline solution was then assessed, followed by sputum induction and, 1 week later, bronchoscopy with bronchoalveolar lavage and bronchial brush biopsy to assess inflammatory cells. RESULTS: Clinical asthma severity was associated with airway hyperresponsiveness. Metachromatic cells were the main granulocyte present in bronchial brush biopsy specimens and correlated with airway responsiveness to saline solution (r = -0.75), methacholine (r = -0.74), peak flow variability (r = 0.59), and clinical asthma severity (r = 0.57). Eosinophils were the main granulocyte present in sputum and correlated with airway responsiveness to saline solution (r = -0.63) but not with other clinical markers of asthma severity. Bronchoalveolar lavage cell counts were not related to clinical asthma severity. CONCLUSIONS: In asthmatic patients treated with cortico-steroids, the dominant inflammatory effector cell in the epithelium is the metachromatic cell, and in sputum it is the eosinophil. These cells correlate with the degree of airway hyperresponsiveness. Clinical asthma severity correlates with airway responsiveness and epithelial metachromatic cells. Induced sputum eosinophils and airway responsiveness to hypertonic saline solution may be useful markers of airway inflammation for clinical practice.     

Activation of the TGF-beta/activin-Smad2 pathway during allergic airway inflammation

Changes in the levels of transforming growth factor (TGF)-beta cytokines or receptors observed during the progression of several inflammatory and fibrotic disorders have been used to implicate these cytokines in the pathophysiology of these diseases. Although correlative, these studies were inconclusive because they were unable to demonstrate actual continuous TGF-beta-mediated signaling in the involved tissues. We reasoned that the phosphorylation state and subcellular localization of Smad2, the intracellular effector of TGF-beta/activin-mediated signaling, could be used as a marker of active signaling mediated by these cytokines in situ. We therefore used an experimental model of ovalbumin-induced allergic airway inflammation and were able to demonstrate a dramatic increase in the numbers of bronchial epithelial, alveolar, and infiltrating inflammatory cells expressing nuclear phosphorylated Smad2 within the allergen-challenged lungs. This was accompanied by strong upregulation of the activin receptor ALK-4/ActR-IB and redistribution of the TGF-beta responsive ALK-5/TbetaR-I. Although levels of TGF-beta1, TGF-beta2, and TGF-beta3 messenger RNA (mRNA) were marginally altered, the level of activin mRNA was strongly upregulated during the inflammatory response. Our data illustrate the usefulness of antiphosphorylated Smad antibodies in demonstrating active TGF- beta/activin-mediated signaling in vivo and strongly suggest that activin/Smad-mediated signaling could be a critical contributor in the pathophysiology of allergic pulmonary diseases.     

Schwann cells genetically modified to express neurotrophins promote spiral ganglion neuron survival in vitro

The intracochlear infusion of neurotrophic factors via a mini-osmotic pump has been shown to prevent deafness-induced spiral ganglion neuron (SGN) degeneration; however, the use of pumps may increase the incidence of infection within the cochlea, making this technique unsuitable for neurotrophin administration in a clinical setting. Cell- and gene-based therapies are potential therapeutic options. This study investigated whether Schwann cells which were genetically modified to over-express the neurotrophins brain-derived neurotrophic factor (BDNF) or neurotrophin 3 (Ntf3, formerly NT-3) could support SGN survival in an in vitro model of deafness. Co-culture of either BDNF over-expressing Schwann cells or Ntf3 over-expressing Schwann cells with SGNs from early postnatal rats significantly enhanced neuronal survival in comparison to both control Schwann cells and conventional recombinant neurotrophin proteins. Transplantation of neurotrophin over-expressing Schwann cells into the cochlea may provide an alternative means of delivering neurotrophic factors to the deaf cochlea for therapeutic purposes.     

Inhaled Cryptococcus neoformans elicits allergic airway inflammation independent of Nuclear Factor Kappa B signalling in lung epithelial cells

Pulmonary challenge with the ubiquitous fungus Cryptococcus neoformans results in allergic airway inflammation (AAI) characterized by robust recruitment of eosinophils and T cells producing type 2 cytokines to the lungs. Previous studies have demonstrated a critical role for Nuclear Factor Kappa B (NF‐κB) activation within lung epithelial cells (LECs) in driving AAI in response to protein allergens, yet the role of LEC‐intrinsic NF‐κB in promoting AAI following exposure to C. neoformans is poorly understood. To investigate the role of LEC‐intrinsic NF‐κB in promoting AAI following C. neoformans challenge, we used IKK^?LEC mice, which lack canonical NF‐κB activation specifically within LECs. IKK^?LEC and littermate control mice were intranasally challenged with 10⁶ CFU of C. neoformans strain 52D, and lung tissues were collected at 7, 14 and 21 days post infection to assess the development of AAI. Notably, the absence of epithelial NF‐κB signalling did not affect the magnitude or kinetics of lung eosinophilia when compared with the response in wild‐type control mice. The total numbers of lung T cells producing the type 2 cytokines interleukin‐5 and interleukin‐13 were also unchanged in IKK^?LEC mice. Furthermore, IKK^?LEC mice showed no defect in the recruitment of protective interferon‐γ‐producing CD4 T cells to the lungs, fungal clearance, or host survival compared with control mice. Immunofluorescence imaging surprisingly revealed no evidence of nuclear localization of NF‐κB in LECs in response to C. neoformans challenge, indicating that NF‐κB is not activated within these cells. Taken together, these data strongly suggest that NF‐κB signalling within LECs does not promote AAI observed in response to C. neoformans.     

Role of eosinophils and their clinical significance in allergic inflammation

Eosinophils are believed to play roles in the pathophysiology of allergic inflammation, such as bronchial asthma. However, recent studies on anti-interleukin-5 monoclonal antibody treatment of asthmatic patients raised the possibility that eosinophils may play only a limited role. More recent studies established that eosinophils are essentially involved in the development of airway remodeling. Moreover, it is theoretically conceivable that eosinophils are a cellular source of lipid mediators, such as cysteinyl leukotrienes or platelet-activating factor in asthma. Even in the absence of interleukin-5, it is likely that the ‘T-helper Type 2 network’, including a cascade of vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, CC chemokines, granulocyte-macrophage colony-stimulating factor, for example, can maintain sufficient eosinophilic infiltration and effector functions, such as superoxide anion generation and degranulation. Long-term studies, wherein tissue eosinophils are eliminated effectively will be required to establish the exact roles of these cells in asthma. Finally, the authors will demonstrate that eosinophils have the potential for not only playing detrimental roles but also beneficial ones.     

IL-4-induced migration of eosinophils in allergic inflammation

T lymphocytes present in allergically inflamed tissue synthesize and secrete the cytokines IL-3, IL-4, IL-5 and GM-CSF which may act as chemotaxins on eosinophils. In contrast to the former cytokines, IL-4 is chemotactic only for eosinophils from peripheral blood of patients with atopic dermatitis and not for eosinophils from normal individuals. IL-4 has the same chemotactic potency as the other cytokines. The optimal chemotactic potency is reached at a concentration of 10 nM. In contrast, neutrophils do not respond chemotactically to IL-4. Checkerboard analysis, inhibition studies with monoclonal anti-IL-4. Abs and desensitization experiments indicated specific interaction of IL-4 with eosinophils. In eosinophils from normal individuals, IL-4 responsiveness could be induced by pretreatment of the cells with IL-5 and GM-CSF. In addition to the fact that IL-4 may be responsible for selective eosinophil transendothelial migration, IL-4 may exert an important modulatory mode of action on eosinophil migration and function within allergically inflamed tissue. Our findings suggest the presence of a functional IL-4R on eosinophils from atopic dermatitis patients.