Betamethasone prevents virus-induced airway inflammation but not airway hyperresponsiveness in guinea pigs

In this study the effect of betamethasone was investigated in guinea pigs that demonstrate airway inflammation and airway hyperresponsiveness after a viral respiratory tract infection with parainfluenza-3 (PI3) virus. Guinea pigs were pretreated with saline or betamethasone 8 mg/kg intraperitoneally twice a day for five consecutive days, starting on day 0 and ending on day 4. On day 1, the guinea pigs were inoculated with either control solution (medium) or PI3 virus. On day 5, airway responsiveness was measured. Furthermore, a blood sample was taken, lungs were lavaged, blood leucocytes were counted, and bronchoalveolar lavage (BAL) cells were counted and differentiated. Accordingly, the activity of the bronchoalveolar cells was measured by lucigenin-amplified chemiluminescence. In virus-infected guinea pigs the total bronchoalveolar cell number was increased by 44% compared with medium-treated guinea pigs. This was mainly due to the increase in macrophages (70%, P  < 0.05) and eosinophils (344%, P  < 0.001). The increase in both total and differential (macrophages and eosinophils) cell numbers in virus-infected guinea pigs was completely abolished in animals treated with betamethasone. Moreover, betamethasone prevented the decrease in number of blood leucocytes in virus-infected guinea pigs. In contrast, betamethasone did not prevent the increase in airway responsiveness to both histamine (>200%) and methacholine (>100%) after the virus infection. In conclusion, betamethasone treatment prevents virus-induced airway inflammation but not airway hyperresponsiveness in guinea pigs.     

Eotaxin and nitric oxide production as markers of inflammation in allergic cynomolgus monkeys

BACKGROUND: Cynomolgus monkeys have a natural hypersensitivity to Ascaris suum antigen. Inhalation of antigen produces immediate and delayed allergic reactions and an influx of inflammatory cells into the lungs. This study investigated the production of nitric oxide (NO) and the chemokine eotaxin during this allergic response. The effect of bronchoscopy alone on lung inflammatory cells was also investigated along with the time course of the eosinophil influx into the lung. METHODS: Allergic cynomolgus monkeys were challenged with antigen. Bronchoalveolar lavage (BAL) was performed before and after challenge, and end-tidal NO was measured before and 24 h after challenge. Eotaxin was measured in the BAL fluid 6, 24 and 72 h after challenge. One group of animals was treated with dexamethasone before challenge to block the influx of cells into the lung. RESULTS: BLA alone induced an influx of neutrophils, but not eosinophils, into the lung 24 h later. A single antigen challenge produced a marked increase in BAL eosinophils that was apparent at 6 h but increased at 72 h after challenge. The increase at 6 h was largely blocked by dexamethasone. Three antigen challenges produced elevated BAL eosinophil levels that persisted for at least 8 weeks. Eotaxin levels rose dramatically 6 h after challenge and remained the same after 24 h. By 72 h, the eotaxin levels had returned to baseline. The increase in eotaxin at 6 h was nonsignificantly reduced by dexamethasone. Exhaled NO levels doubled 24 h after challenge and were not affected by dexamethasone. CONCLUSIONS: Eotaxin and NO production were increased after airway challenge in allergic monkeys. The rise in NO was not blocked by dexamethasone. The effects of bronchoscopy on the BAL can be avoided by using alternate lungs on consecutive occasions. Eosinophils persist in the BAL for many weeks after antigen challenge.     

Eotaxin expression in Sephadex-induced lung injury in rats

The CC chemokine eotaxin is a potent and specific eosinophil chemoattractant. Eosinophil-dependent tissue injury has been shown to contribute to airway inflammation such as that in asthma. In the present study, We investigated eotaxin expression in a rat model of pulmonary inflammation (featuring accumulation of eosinophils) induced by intratracheal instillation of cross-linked dextran beads (Sephadex G200). Intratracheal instillation of 5 mg/kg Sephadex caused a time-dependent eosinophil infiltration into the lung, reaching a peak at 24 hours. Eotaxin mRNA in the lung paralleled the eosinophil influx. Eotaxin protein in bronchoalveolar (BAL) fluids and lung homogenates was shown by Western blot and immunostaining to be maximally expressed by 24 hours. Sephadex-induced lung injury, as measured by (125)I-labeled albumin leakage from the pulmonary vasculature, developed in a time-dependent manner. Intravenous injection of blocking antibody to eotaxin significantly decreased eosinophil infiltration and lung permeability. These data suggest that, in the Sephadex model of lung inflammation, eotaxin up-regulation mediates intrapulmonary accumulation of eosinophils and the development of lung injury.     

alpha4 integrin-dependent eotaxin induction of bronchial hyperresponsiveness and eosinophil migration in interleukin-5 transgenic mice

We investigated the roles of eosinophil infiltration and activation induced by the eosinophil-selective chemokine eotaxin, and of the expression of eosinophil alpha4 and beta2 integrins in causing bronchial hyperresponsiveness (BHR) in interleukin (IL)-5 CBA/Ca transgenic mice. These mice did not show BHR, despite the presence of some eosinophils in the lungs. Intratracheal mouse recombinant eotaxin (3 micrograms) did not induce BHR in wild-type mice. In IL-5 transgenic mice, eotaxin (3 and 5 micrograms) increased responsiveness at 24 h and increased eosinophils in bronchoalveolar lavage (BAL) fluid by 9.4- and 14-fold by 24 h, respectively, together with augmentation of eosinophil peroxidase activity and eosinophil infiltration in the airway submucosa. Using flow cytometry, the expression of alpha4, CD11b, and CD18 was upregulated in BAL, but not in blood, eosinophils. A rat anti-alpha4 antibody inhibited eotaxin-induced BHR and eosinophil migration and activation, but an anti-CD11b antibody had no significant effects on BHR. A combination of both antibodies was more effective. IL-5 and eotaxin synergize in the induction of BHR and airway eosinophilia, effects that are dependent on the induction of eosinophil alpha4 integrin. Expression of BHR depends on the recruitment and activation of eosinophils.     

Expression of eotaxin in induced sputum of atopic and nonatopic asthmatics

BACKGROUND: The chemokine eotaxin has been implicated in airway eosinophilia in atopic asthma. We have compared airway eosinophils and eotaxin expression in induced sputum from well-matched atopic and nonatopic asthmatics. METHODS: Eosinophil numbers, eosinophil cationic protein (ECP), and the expression of eotaxin were examined in induced sputum from atopic asthmatics (AA = 11), nonatopic asthmatics (NAA = 11), and atopic (AC = 12) and normal (NC = 10) controls. Slides were prepared for differential cell counts by Romanowsky stain, and ECP levels were measured by RIA. Eotaxin expression was detected by in situ hybridization, with 35S-labelled riboprobes and immunocytochemistry. RESULTS: The numbers of eosinophils and ECP concentration were increased in the sputum of AA and NAA compared with AC and NC (P < 0.05). The numbers of eotaxin mRNA+ and immunoreactive cells were increased in NAA, but not AA, when compared with controls (P < 0.05). Eotaxin immunoreactive cells in NAA were significantly higher than in AA (P < 0.05). Eotaxin was expressed predominantly by macrophages, eosinophils, and epithelial cells. In NAA, but not AA, the numbers of eotaxin mRNA+ cells were correlated with histamine PC20 (r = -0.81, P < 0.01) and eosinophil numbers in sputum (r = 0.7, P < 0.05). CONCLUSIONS: Eotaxin production by macrophages, eosinophils, and epithelial cells may play a more pronounced role in airway eosinophilia in nonatopic than in atopic asthma.     

Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma. Association with airway hyperresponsiveness and predominant co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells

Eotaxin is a newly discovered C-C chemokine which preferentially attracts and activates eosinophil leukocytes by acting specifically on its receptor CCR3. The airway inflammation characteristic of asthma is believed to be, at least in part, the result of eosinophil-dependent tissue injury. This study was designed to determine whether there is increased expression of eotaxin and CCR3 in the bronchial mucosa of asthmatics and whether this is associated with disease severity. The major sources of eotaxin and CCR3 mRNA were determined by co-localization experiments. Bronchial mucosal biopsy samples were obtained from atopic asthmatics and normal non-atopic controls. Eotaxin and CCR3 mRNA were identified in tissue sections by in situ hybridization (ISH) using radiolabeled riboprobes and their protein product visualized by immunohistochemistry (IHC). Co-localization experiments were performed by double ISH/IHC. Eotaxin and CCR3 (mRNA and protein) were significantly elevated in atopic asthmatics compared with normal controls. In the asthmatics there was a highly significant inverse correlation between eotaxin mRNA⁺ cells and the histamine provocative concentration causing a 20% fall in FEV₁ (PC₂₀). Cytokeratin-positive epithelial cells and CD31⁺ endothelial cells were the major source of eotaxin mRNA whereas CCR3 co-localized predominantly to eosinophils. These data are consistent with the hypothesis that damage to the bronchial mucosa in asthma involves secretion of eotaxin by epithelial and endothelial cells resulting in eosinophil infiltration mediated via CCR3. Since selective (eotaxin) and non-selective C-C chemokines such as RANTES, MCP-3 and MCP-4 all stimulate eosinophils via CCR3, this receptor is potentially a prime therapeutic target in the spectrum of diseases involving eosinophil-mediated tissue damage.     

The onset and recovery from airway hyperresponsiveness: relationship with inflammatory cell infiltrates and release of cytotoxic granule proteins

Previous studies from our laboratory have demonstrated a temporal relationship between eosinophil influx into the airways and the onset of airway hyperresponsiveness to inhaled methacholine. The purpose of the present study was to extend this observation by evaluating changes in airway cellular composition and measuring the levels of granulocyte-derived mediators recovered in BAL fluid during the onset and recovery from antigen-induced airway hyperresponsiveness. Airway cellular composition, airway responsiveness to inhaled methacholine and the levels of BAL fluid EPO and MPO were monitored over a 32 day study in eight adult male Ascaris suum sensitive cynomolgus monkeys. Repeated Ascaris suum inhalation (nine challenges during days 0-21) resulted in a selective, sustained airway eosinophilia that was temporally related with the onset and maintenance of airway hyperresponsiveness (r = 0.67, P less than 0.001). The level of BAL eosinophil-derived EPO was increased and remained elevated concurrent with the increase in airway eosinophils and airway responsiveness. During the recovery phase (days 22-32) the actual number of eosinophils remained elevated, while BAL EPO levels were significantly decreased. The recovery phase was also associated with a transient increase in the number of BAL neutrophils and MPO concentration. We conclude that the number and state of activation of airway eosinophils directly correlate with the onset and maintenance of airway hyperresponsiveness. Recovery from airway hyperresponsiveness is associated with a decrease in eosinophil activation and a transient increase in the number of activated neutrophils.     

Role of Airway Eosinophilia and Eosinophil Activation in Sephadex-induced Inflammation

The effects of an intravenous injection of Sephadex beads on lung eosinophil infiltration and eosinophil peroxydase activity and its relationship to bronchial hyperresponsiveness was examined in guinea pigs. This Sephadex beads injection led to blood, lung and airway eosinophilia in association with bronchial hyperresponsiveness. Histologic examination of the lower bronchus indicated that the eosinophil number increased markedly in the mucosa and submucosa. In addition, the eosinophils surrounding the bronchioles 1 day after the Sephadex injection migrated further in airway submucosa and mucosa 7 and 14 days after. Moreover, the bronchial hyperresponsiveness is observed without histologic evidence of airway epithelium damage. Therefore, the bronchial hyperresponsiveness seems to be more related to the eosinophil infiltration in the airway epithelium and possibly eosinophil activation rather than to the eosinophil number recovered in the BAL fluid. We conclude that the maintenance of hyperresponsiveness state could be associated with the persistence of blood and airway eosinophilia.     

Experimental common cold increases mucosal output of eotaxin in atopic individuals

BACKGROUND: In view of recent observations demonstrating that rhinovirus infections are associated with increased local activity of eosinophils, we hypothesized that eotaxin, a selective eosinophil chemoattractant, may be involved in eosinophil recruitment/activation in common cold infections. METHODS: Twenty-three patients with seasonal allergic rhinitis were inoculated with human rhinovirus 16 (HRV 16) outside the pollen season. Sequential diluent and allergen (10(3) SQ-U) challenges, each followed by nasal lavage, were carried out about 3 months before and 4 days after virus inoculation. RESULTS: Seventeen patients developed common cold and were further analyzed. Lavage fluid levels of eotaxin were increased at the height of active common cold (day 4 after inoculation) both at baseline (after diluent challenge) (P<0.01) and acutely after allergen challenge (P<0.05). CONCLUSIONS: Common cold symptoms after nasal HRV 16 inoculation are associated with increased nasal mucosal output of eotaxin, suggesting that this chemokine is partly responsible for recruitment/activation of airway mucosal eosinophils in common cold infections.     

Anti-inflammatory actions of interleukin-13: suppression of tumor necrosis factor-alpha and antigen-induced leukocyte accumulation in the guinea pig lung

The Th2 cytokine interleukin (IL)-13 is believed to play an important role in the development of allergy, although it has also been ascribed anti-inflammatory roles in several experimental models. In this study, we have examined the effects of human recombinant IL-13 on eosinophilic lung inflammation in the guinea pig. IL-13 (1 to 100 ng, given by intratracheal instillation) did not elicit airway eosinophil recruitment. A pronounced accumulation of eosinophils, as well as monocyte/macrophages, was elicited by intratracheal instillation of guinea pig tumor necrosis factor alpha (gpTNF-alpha). Intratracheal administration of IL-13 (1 to 100 ng) given immediately prior to exposure to gpTNF-alpha resulted in a dose-related suppression of eosinophil and monocyte/macrophage accumulation in the airways, as assessed by bronchoalveolar lavage (BAL) and eosinophil peroxidase activity in whole-lung homogenates. IL-13 treatment also reduced BAL fluid (BALF) leukocyte accumulation induced by subsequent aerosol antigen challenge of sensitized guinea pigs. Antigen challenge also resulted in elevated levels of immunoreactive eotaxin and eosinophil-stimulating activity in BALF, although only the latter was reduced significantly by IL-13 instillation prior to challenge. In contrast to the suppressive effects of IL-13, instillation of human recombinant IL-4 (100 ng) alone elicited an increase in BALF monocyte/macrophage numbers, and IL-4 was unable to inhibit gpTNF-alpha-induced leukocyte accumulation. Hence, IL-13 (but not human IL-4) exhibits an anti-inflammatory action in the airways of gpTNF-alpha- or antigen-challenged guinea pigs, by mechanisms that may involve the decreased generation of eosinophil-stimulating activity in the airways.     

IL-1 receptors mediate persistent, but not acute, airway hyperreactivity to ozone in guinea pigs

Ozone exposure in the lab and environment causes airway hyperreactivity lasting at least 3 days in humans and animals. In guinea pigs 1 day after ozone exposure, airway hyperreactivity is mediated by eosinophils that block neuronal M(2) muscarinic receptor function, thus increasing acetylcholine release from airway parasympathetic nerves. However, mechanisms of ozone-induced airway hyperreactivity change over time, so that depleting eosinophils 3 days after ozone makes airway hyperreactivity worse rather than better. Ozone exposure increases IL-1beta in bone marrow, which may contribute to acute and chronic airway hyperreactivity. To test whether IL-1beta mediates ozone-induced airway hyperreactivity 1 and 3 days after ozone exposure, guinea pigs were pretreated with an IL-1 receptor antagonist (anakinra, 30 mg/kg, intraperitoneally) 30 minutes before exposure to filtered air or to ozone (2 ppm, 4 h). One or three days after exposure, airway reactivity was measured in anesthetized guinea pigs. The IL-1 receptor antagonist prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone exposure. Ozone-induced airway hyperreactivity was vagally mediated, since bronchoconstriction induced by intravenous acetylcholine was not changed by ozone. The IL-1 receptor antagonist selectively prevented ozone-induced reduction of eosinophils around nerves and prevented ozone-induced deposition of extracellular eosinophil major basic protein in airways. These data demonstrate that IL-1 mediates ozone-induced airway hyperreactivity at 3 days, but not 1 day, after ozone exposure. Furthermore, preventing hyperreactivity was accompanied by decreased eosinophil major basic protein deposition within the lung, suggesting that IL-1 affects eosinophil activation 3 days after ozone exposure.     

Association of interleukin-5 and eotaxin with acute exacerbation of asthma

BACKGROUND: Airway eosinophilia is frequently observed during acute exacerbation of asthma. Interleukin-5 (IL-5) and eotaxin are directly involved in the airway eosinophilia found in persistent asthma. Interrelation between these cytokines is expected to occur in acute exacerbation of asthma. Thus, we evaluated the relevance of interaction between eotaxin and IL-5 in the airway inflammation of acute exacerbation. METHODS: We measured the number of inflammatory cells and the amount of eotaxin and IL-5 in sputum from 22 healthy subjects, 21 asthmatics with acute exacerbation and 16 patients with mild persistent asthma, and reassessed these values in 7 subjects with acute exacerbation after 7 days' treatment with systemic steroid (2 mg/kg/day). Sources of IL-5 and eotaxin were investigated by immunohistochemical staining of sputum cells of 4 cases from each group. RESULTS: Both IL-5 and eotaxin levels were higher in patients with acute exacerbation of asthma than in patients with persistent asthma and normal subjects. IL-5 and eotaxin levels were significantly correlated with eosinophil percentages in mild persistent asthma. Eotaxin staining was found mainly on macrophages and occasionally on eosinophils. Steroid treatment markedly decreased eosinophil percentages and IL-5 levels within 7 days but did not alter eotaxin levels. CONCLUSIONS: Both IL-5 and eotaxin are associated with acute exacerbation of asthma. IL-5 rather than eotaxin is effectively decreased by the inhibitory effect of steroid in acute exacerbation.     

Differential expression of CCR3 ligand mRNA in guinea pig lungs during allergen-induced inflammation

OBJECTIVE AND DESIGN: The gene expression profile of CCR3 ligands, eotaxin, RANTES, and monocyte chemoattractant protein-3 (MCP-3), was examined in normal and inflamed guinea pig lungs. MATERIAL: Male Hartley guinea pigs (n = 49). METHODS: Pulmonary mRNA was obtained from naive animals, animals treated with intravenous lipopolysaccharide administration, and animals repeatedly exposed to aerosolized allergen (ovalbumin). Northern analysis was performed to quantify pulmonary expression of eotaxin, RANTES, and MCP-3 mRNA. Pulmonary eosinophil peroxidase (EPO) activity was measured to quantify eosinophil accumulation. RESULTS: Eotaxin and RANTES mRNAs, but not MCP-3 mRNA, were constitutively expressed in guinea pig lungs. Lipopolysaccharide treatment increased MCP-3 mRNA expression, but not eotaxin or RANTES mRNA. In contrast, allergen exposure in sensitized animals caused an increase in eotaxin mRNA, which demonstrated good temporal and quantitative correlation with pulmonary EPO activity, but not in MCP-3 or RANTES mRNA. CONCLUSIONS: Guinea pig CCR3 ligands demonstrated different gene expression profiles in normal and inflamed airways, suggesting that they play different physiological and pathophysiological roles in the airway.     

Influenza virus A stimulates expression of eotaxin by nasal epithelial cells

BACKGROUND: Respiratory virus is one of the most common causes of airway inflammation, but its pathogenic mechanisms are not well understood. Eotaxin is a potent eosinophil chemoattractant and is a selective agonist for C-C chemokine receptor 3 (CCR3). Although it has recently been demonstrated that epithelial cells express eotaxin, both in vivo and in vitro, there are few data concerning the expression in viral infection. OBJECTS: We hypothesized that eotaxin may play an important role in attracting inflammatory cells into the airway after viral infection and analysed whether viral infection induces eotaxin in nasal epithelial cells in vitro. METHODS: Nasal epithelial cells obtained from polypectomy for nasal polyp were infected with influenza virus A (subtype H3N2). The cells and supernatants were collected 8, 24 and 48 h after infection. Eotaxin mRNA was analysed by RT-PCR. Eotaxin concentration in the supernatants was analysed by enzyme-linked immunosorbent assay. We also examined a blocking assay to analyse the intervention of pro-inflammatory cytokines, TNF-alpha and IL-1beta in eotaxin production induced by influenza virus. RESULTS: The results showed that eotaxin was expressed constitutively in uninfected cells, but was up-regulated for both mRNA and protein levels in infected cells. Blocking experiments using anti-TNF-alpha and anti-IL-1beta antibodies showed no effects of these agents on the level of eotaxin. In addition, UV-inactivated virus did not enhance the expression of eotaxin. CONCLUSIONS: These results suggest that influenza virus A infection in nasal epithelial cells stimulates the expression of eotaxin, and may play an important role in the pathogenesis of airway inflammation by inducing eotaxin.     

Characterization of airway inflammation after repeated exposures to occupational agents

BACKGROUND: Little is known about the comparative kinetics of eosinophil recruitment after exposure to low- and high-molecular-weight sensitizers in subjects with occupational asthma (OA). OBJECTIVES: The aims of the study were to investigate the kinetics of changes in inflammatory mediators associated with eosinophil infiltration (IL-5 and eotaxin) and to examine the nature of the airway inflammation induced in response to different types of occupational agents. METHODS: We investigated 15 subjects with OA caused by high- and low-molecular-weight agents. The subjects were exposed to increasing doses of the relevant occupational agent over 3 to 4 days until a 20% fall in FEV(1) occurred. Methacholine challenge and sputum induction were performed at the end of each day of exposure. Sputum samples were assessed for differential cell counts, including eosinophils, IL-5, and eotaxin messenger RNA. RESULTS: There was an increase in sputum eosinophils, eotaxin, and IL-5 on the day preceding the occurrence of asthmatic reaction, although there was no change in functional parameters (FEV(1) and PC(20)). Increase in sputum eosinophils was more prominent in subjects exposed to low-molecular-weight agents than to high-molecular-weight agents. CONCLUSION: Changes in eosinophils, IL-5, and eotaxin precede functional changes after exposure to occupational agents in subjects with OA. Eosinophil inflammation is a feature of exposure to both high- and low-molecular-weight agents. Induced sputum may be a useful tool in the early diagnosis of OA.     

Mucosal output of eotaxin in allergic rhinitis and its attenuation by topical glucocorticosteroid treatment

BACKGROUND: Eotaxin is a chemokine that attracts and activates eosinophils. The present study examines the occurrence of eotaxin in nasal mucosal surface liquids in patients with seasonal allergic rhinitis without allergen exposure and during repeat allergen challenge with and without topical glucocorticosteroid treatment. The number of subepithelial eosinophils and mucosal outputs of bulk plasma (alpha2-macroglobulin) and eosinophil cationic protein (ECP) are also examined. METHODS: Twelve patients underwent daily allergen challenges for 6 days. Separately, 14 patients, who were receiving budesonide and placebo in a parallel group design, also underwent allergen challenge for 6 days. Nasal biopsies were obtained before and 24 h after the allergen challenge series, and lavages were carried out before and 15 min after selected allergen challenges. RESULTS: At baseline nasal lavage fluid levels of eotaxin correlated to levels of alpha2-macroglobulin and ECP. After the first allergen challenge there was a correlation between nasal lavage fluid levels of eotaxin and ECP. Repeat allergen exposure increased the mucosal output of eotaxin (P <0.05) and ECP (P <0.01) as well as eosinophil numbers (P <0.01), but no correlation was found between increased eosinophil numbers and eotaxin. Budesonide reduced eotaxin levels during repeat allergen challenge (P <0.05). CONCLUSIONS: Repeat allergen exposure in allergic rhinitis is associated with increased mucosal output of eotaxin. Topical budesonide attenuates this effect, suggesting the possibility that inhibitory effects on mucosal eotaxin may contribute to anti-eosinophilic actions of topical glucocorticosteroids.     

Increases in eotaxin‐positive cells in induced sputum from atopic asthmatic subjects after inhalational allergen challenge

BACKGROUND: Eosinophils are believed to be critical proinflammatory cells in airway mucosal damage in asthma. Eotaxin is a C-C chemokine with selective activity for eosinophils and basophils. Previous studies have shown increased expression of eotaxin in the airways of asthmatics at baseline. We aimed to investigate eotaxin expression during the late-phase reaction to allergen inhalation in atopic asthmatics. METHODS: Sputum induction was performed before and 24 h after inhalational allergen challenge in atopic asthmatics, and eotaxin protein was detected immunocytochemically. RESULTS: Thirteen patients with a mean decrease in forced expiratory volume in 1 s of 28% (+/-1.5) during the early asthmatic reaction, and 39% (+/-4.7) during the late asthmatic reaction produced sufficient sputum for study. The percentage of eosinophils in sputum was increased 24 h after allergen challenge (P<0.004), and eosinophil percentages in sputum after challenge correlated with the magnitude of the late-phase reaction (r=0.56, P=0.05). The percentage of eotaxin-positive cells increased from 12.6% (range 2-43.8) to 24.3% (8.1-47.1, P<0.005). Allergen-induced increases in eotaxin-positive cells correlated with increases in eosinophils (r=0.63, P<0.01). CONCLUSIONS: These findings suggest that eotaxin may contribute to allergen-induced recruitment of eosinophils to the airway in asthmatic subjects.     

Eosinophil activation by eotaxin--eotaxin primes the production of reactive oxygen species from eosinophils

BACKGROUND: The CC chemokine eotaxin has been shown to possess selective chemotactic activity for eosinophils, the major effector cells in allergic inflammation. Reactive oxygen species (ROS) from eosinophils may damage cells or tissue, such as the mucosal epithelium. In this study, we examined the effect of eotaxin on ROS from eosinophils and compared its activity with RANTES and interleukin (IL)-5. Moreover, we examined the signal transduction of eotaxin and the effect of dexamethasone on ROS from eosinophils. METHODS: Eosinophils were isolated by modified CD16-negative selection. ROS in luminol-dependent or lucigenin-dependent chemiluminescence reaction were examined. Calcium ionophore A23187 was added to the mixture of eosinophils with luminol or lucigenin, and then ROS were determined. RESULTS: Eotaxin primed the production of ROS in a dose-dependent manner. ROS from untreated eosinophils evoked with calcium ionophore A23187 in luminol-dependent chemiluminescence gave a maximal value of 4957+/-1035 intensity counts (IC) (mean+/-SE, n=7) and an integral value of 15.75+/-3.14 IC (x10(-4)), while eosinophils that were treated with eotaxin gave maximal values of 11 142+/-2300 IC (10 nM) and 29165+/-3718 IC (100 nM) and integral values of 41.07+5.44 IC (x10(-4)) (10 nM) and 152.90+/-22.38 IC (x10(-4))(100 nM). Moreover, eotaxin was less effective as a priming agent with lucigenin-sensitive pathways than luminol-sensitive pathways. Among several kinds of eosinophils activating cytokines and chemokines, the priming effect of eotaxin on RO5 was the most potent. Eotaxin-primed ROS were inhibited by pertussis toxin, which ADP-ribolysates G proteins; wortmannin, a phosphatidylinositol-3-kinase inhibitor; and genistein, a tyrosine kinase inhibitor, suggesting the involvement of pertussis toxin-sensitive G proteins, phosphatidylinositol-3-kinase, and tyrosine kinase in the signal transduction of eotaxin. Moreover, dexamethasone inhibited ROS from not only untreated eosinophils but also eosinophils treated with eotaxin. CONCLUSION: Eotaxin may play an important role in the pathogenesis of allergic inflammation through eosinophil activation by priming of eosinophil oxidative metabolism, as well as by involvement in selective eosinophil chemotaxis.     

Airway eosinophilic inflammation, epithelial damage, and bronchial hyperresponsiveness in patients with mild–moderate, stable asthma

Allergic asthma is characterized by chronic recruitment of eosinophils in the airways. Once activated, eosinophils release toxic products, including eosinophil cationic protein (ECP), able to damage airway epithelial cells. To test the hypothesis that also in mild-moderate stable asthma, a significant eosinophil activation could occur, we studied 25 asthmatic patients (34 ± 19 years old), of whom 18 were allergic (27 ± 12 years) and seven nonallergic (42±10 years), with FEV₁ values ±70% of predicted, and eight normal volunteers (controls, 33 ±11 years). All subjects underwent methacholine (MCh) challenge on the first visit, and bronchoalveolar lavage (BAL) on the second visit (approximately 3–4 days later). BAL cells were counted and albumin (Alb) (as index of protein dilution in BAL fluid) and ECP levels (as index of eosinophil activation) in BAL fluid were measured. As compared to controls, a significant increase in BAL eosinophil and in BAL epithelial cell numbers was observed in asthmatic patients (P>0.05, each comparison), with no differences between the two asthmatic patient subgroups. Detectable ECP levels (>2 μg/1) were found in BAL of 18 asthmatic patients (14 allergic and four nonallergic asthmatic patients), while Alb levels were measurable in 25 BAL fluids and found to be similar in controls and asthmatic patients, and in the two asthmatic patient subgroups (P>0.05, each comparison). In BAL of asthmatic patients, positive correlations were found between eosinophil numbers and 1) ECP/Alb levels (r= 0.50, P = 0.020); 2) epithelial cell numbers (r = 0.S0, P = 0.014). In asthmatic patients, a significant negative correlation was found between bronchial reactivity to MCh (log Pd₁₅) and ECP/Alb levels in BAL fluid (r=-0.6, P= 0.005), whereas no correlation was found between log Pd₁₅ MCh and BAL eosinophil or epithelial cell number (P>0.1, each correlation). These data suggest that bronchial eosinophil recruitment and activation may occur also in mild-moderate stable asthma and that bronchial epithelium damage and airway responsiveness may be partially associated with the eosinophilic inflammatory reaction.