Number Of Eosinophils In Bronchoalveolar Lavage Research Articles

Bovine Serum Albumin Elicits IL-33-Dependent Adipose Tissue Eosinophilia: Potential Relevance to Ovalbumin-induced Models of Allergic Disease.

All cells of the immune system reside in adipose tissue (AT), and increasing type 2 immune cells may be a therapeutic strategy to improve metabolic health. In our previous study using i.p. IL-5 injections to increase eosinophils, we observed that a standard vehicle control of 0.1% BSA also elicited profound AT eosinophilia. In this study, we aimed to determine whether BSA-induced AT eosinophilia results in metabolic benefits in murine models of diet-induced obesity. I.p. 0.1% BSA injections increased AT eosinophils after 4 wk. Despite elevating eosinophils to >50% of immune cells in the AT, body weight and glucose tolerance were not different between groups. Interestingly, BSA elicited epithelial IL-33 production, as well as gene expression for type 2 cytokines and IgE production that were dependent on IL-33. Moreover, multiple models of OVA sensitization also drove AT eosinophilia. Following transplantation of a donor fat pad with BSA-induced eosinophilia, OVA-sensitized recipient mice had higher numbers of bronchoalveolar lavage eosinophils that were recipient derived. Interestingly, lungs of recipient mice contained eosinophils, macrophages, and CD8 T cells from the donor AT. These trafficked similarly from BSA- and non-BSA-treated AT, suggesting even otherwise healthy AT serves as a reservoir of immune cells capable of migrating to the lungs. In conclusion, our studies suggest that i.p. injections of BSA and OVA induce an allergic response in the AT that elicits eosinophil recruitment, which may be an important consideration for those using OVA in animal models of allergic disease.

Early-life heterologous rhinovirus infections induce an exaggerated asthma-like phenotype

Early-life wheezing-associated respiratory tract infection by rhinovirus (RV) is a risk factor for asthma development. Infants are infected with many different RV strains per year. We previously showed that RV infection of 6-day-old BALB/c mice induces a mucous metaplasia phenotype that is dependent on type 2 innate lymphoid cells (ILC2s). We hypothesized that early-life RV infection alters the response to subsequent heterologous infection, inducing an exaggerated asthma-like phenotype. Wild-type BALB/c mice and Rorafl/flIl7rcre mice lacking ILC2s were treated as follows: (1) sham on day 6 of life plus sham on day 13 of life, (2) RV-A1B on day 6 plus sham on day 13, (3) sham on day 6 plus RV-A2 on day 13, and (4) RV-A1B on day 6 plus RV-A2 on day 13. Mice infected with RV-A1B at day 6 and sham at day 13 showed an increased number of bronchoalveolar lavage eosinophils and increased expression of IL-13 mRNA but not expression of IFN-γ mRNA (which is indicative of a type 2 immune response), whereas mice infected with sham on day 6 and RV-A2 on day 13 of life demonstrated increased IFN-γ expression (which is a mature antiviral response). In contrast, mice infected with RV-A1B on day 6 before RV-A2 infection on day 13 showed increased expression of IL-13, IL-5, Gob5, Muc5b, and Muc5ac mRNA; increased numbers of eosinophils and IL-13-producing ILC2s; and exaggerated mucus metaplasia and airway hyperresponsiveness. Compared with Rorafl/fl mice, Rorafl/flIl7rcre mice showed complete suppression of bronchoalveolar lavage eosinophils and mucous metaplasia. Early-life RV infection alters the response to subsequent heterologous infection, inducing an intensified asthma-like phenotype that is dependent on ILC2s.

Aqueous extracts from Uncaria tomentosa (Willd. ex Schult.) DC. reduce bronchial hyperresponsiveness and inflammation in a murine model of asthma

Ethnopharmacological relevanceUncaria tomentosa (Willd. Ex Schult) DC is used by indigenous tribes in the Amazonian region of Central and South America to treat inflammation, allergies and asthma. The therapeutic properties of U. tomentosa have been attributed to the presence of tetracyclic and pentacyclic oxindole alkaloids and to phenolic acids. Aims of the studyTo characterize aqueous bark extracts (ABE) and aqueous leaf extracts (ALE) of U. tomentosa and to compare their anti-inflammatory effects. Materials and methodsConstituents of the extracts were identified by ultra performance liquid chromatography-mass spectrometry. Anti-inflammatory activities were assessed in vitro by exposing lipopolysaccharide-stimulated macrophage cells (RAW264.7-Luc) to ABE, ALE and standard mitraphylline. In vivo assays were performed using a murine model of ovalbumin (OVA)-induced asthma. OVA-sensitized animals were treated with ABE or ALE while controls received dexamethasone or saline solution. Bronchial hyperresponsiveness, production of Th1 and Th2 cytokines, total and differential counts of inflammatory cells in the bronchoalveolar lavage (BAL) and lung tissue were determined. ResultsMitraphylline, isomitraphylline, chlorogenic acid and quinic acid were detected in both extracts, while isorhyncophylline and rutin were detected only in ALE. ABE, ALE and mitraphylline inhibited the transcription of nuclear factor kappa-B in cell cultures, ALE and mitraphylline reduced the production of interleukin (IL)−6, and mitraphylline reduced production of tumor necrosis factor-alpha. Treatment with ABE and ALE at 50 and 200 mg kg−1, respectively, reduced respiratory elastance and tissue damping and elastance. ABE and ALE reduced the number of eosinophils in BAL, while ALE at 200 mg kg−1 reduced the levels of IL-4 and IL-5 in the lung homogenate. Peribronchial inflammation was significantly reduced by treatment with ABE and ALE at 50 and 100 mg kg−1 respectively. ConclusionThe results clarify for the first time the anti-inflammatory activity of U. tomentosa in a murine model of asthma. Although ABE and ALE exhibited distinct chemical compositions, both extracts inhibited the production of pro-inflammatory cytokines in vitro. In vivo assays revealed that ABE was more effective in treating asthmatic inflammation while ALE was more successful in controlling respiratory mechanics. Both extracts may have promising applications in the phytotherapy of allergic asthma.

Therapeutic Fluorescent Hybrid Nanoparticles for Traceable Delivery of Glucocorticoids to Inflammatory Sites.

Treatment of inflammatory disorders with glucocorticoids (GCs) is often accompanied by severe adverse effects. Application of GCs via nanoparticles (NPs), especially those using simple formulations, could possibly improve their delivery to sites of inflammation and therefore their efficacy, minimising the required dose and thus reducing side effects. Here, we present the evaluation of NPs composed of GC betamethasone phosphate (BMP) and the fluorescent dye DY-647 (BMP-IOH-NPs) for improved treatment of inflammation with simultaneous in vivo monitoring of NP delivery.Methods: BMP-IOH-NP uptake by MH-S macrophages was analysed by fluorescence and electron microscopy. Lipopolysaccharide (LPS)-stimulated cells were treated for 48 h with BMP-IOH-NPs (1×10-5-1×10-9 M), BMP or dexamethasone (Dexa). Drug efficacy was assessed by measurement of interleukin 6. Mice with Zymosan-A-induced paw inflammation were intraperitoneally treated with BMP-IOH-NPs (10 mg/kg) and mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI) were treated intranasally with BMP-IOH-NPs, BMP or Dexa (each 2.5 mg/kg). Efficacy was assessed in vivo by paw volume measurements with µCT and ex vivo by measurement of paw weight for Zymosan-A-treated mice, or in the AAI model by in vivo x-ray-based lung function assessment and by cell counts in the bronchoalveolar lavage (BAL) fluid and histology. Delivery of BMP-IOH-NPs to the lungs of AAI mice was monitored by in vivo optical imaging and by fluorescence microscopy.Results: Uptake of BMP-IOH-NPs by MH-S cells was observed during the first 10 min of incubation, with the NP load increasing over time. The anti-inflammatory effect of BMP-IOH-NPs in vitro was dose dependent and higher than that of Dexa or free BMP, confirming efficient release of the drug. In vivo, Zymosan-A-induced paw inflammation was significantly reduced in mice treated with BMP-IOH-NPs. AAI mice that received BMP-IOH-NPs or Dexa but not BMP revealed significantly decreased eosinophil numbers in BALs and reduced immune cell infiltration in lungs. Correspondingly, lung function parameters, which were strongly affected in non-treated AAI mice, were unaffected in AAI mice treated with BMP-IOH-NPs and resembled those of healthy animals. Accumulation of BMP-IOH-NPs within the lungs of AAI mice was detectable by optical imaging for at least 4 h in vivo, where they were preferentially taken up by peribronchial and alveolar M2 macrophages.Conclusion: Our results show that BMP-IOH-NPs can effectively be applied in therapy of inflammatory diseases with at least equal efficacy as the gold standard Dexa, while their delivery can be simultaneously tracked in vivo by fluorescence imaging. BMP-IOH-NPs thus have the potential to reach clinical applications.

Protective effect of a Protein Epitope Mimetic CCR10 antagonist, POL7085, in a model of allergic eosinophilic airway inflammation.

BackgroundPotential involvement of the CCR10/CCL28 axis was recently reported in murine models of allergic asthma. If confirmed, blockade of the CCR10 receptor would represent an alternative to current asthma therapies. We evaluated the effect of a novel Protein Epitope Mimetic CCR10 antagonist, POL7085, in a murine model of allergic eosinophilic airway inflammation.MethodsMice were sensitized and challenged to ovalbumin. POL7085, a CCR10 antagonist (7.5 and 15 mg/kg), dexamethasone (1 mg/kg) or vehicle were administered intranasally once daily 1h before each allergen challenge. On day 21, airway hyperresponsiveness, bronchoalveolar lavage inflammatory cells and Th2 cytokines, and lung tissue mucus and collagen were measured.ResultsAllergen challenge induced airway hyperresponsiveness in vehicle-treated animals as measured by whole body barometric plethysmography, and eosinophilia in bronchoalveolar lavage. POL7085 dose-dependently and significantly decreased airway hyperresponsiveness (34 ± 16 %) and eosinophil numbers in bronchoalveolar lavage (66 ± 6 %). In addition, the highest dose of POL7085 used significantly inhibited lung IL-4 (85 ± 4 %), IL-5 (87 ± 2 %) and IL-13 (190 ± 19 %) levels, and lung collagen (43 ± 11 %).ConclusionsThe Protein Epitope Mimetic CCR10 antagonist, POL7085, significantly and dose-dependently decreased allergen-induced airway hyperresponsiveness and airway inflammation after once daily local treatment. Our data give strong support for further investigations with CCR10 antagonists in asthmatic disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0231-5) contains supplementary material, which is available to authorized users.

KCa3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma

BackgroundThe Ca2+-activated K+ channel KCa3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of KCa3.1 modifies experimental asthma in sheep.Methodology and Principal FindingsAtopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the KCa3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.ConclusionsThese findings suggest that KCa3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of KCa3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.

Integrin Activation States and Eosinophil Recruitment in Asthma

Eosinophil arrest and recruitment to the airway in asthma are mediated, at least in part, by integrins. Eosinophils express α4β1, α6β1, αLβ2, αMβ2, αXβ2, αDβ2, and α4β7 integrins, which interact with counter-receptors on other cells or ligands in the extracellular matrix. Whether a given integrin-ligand pair mediates cell adhesion and migration depends on the activation state of the integrin. Integrins exist in an inactive bent, an intermediate-activity extended closed, and a high-activity extended open conformation. Integrin activation states can be monitored by conformation-specific monoclonal antibodies (mAbs). Studies in mice indicate that both β1 and β2 integrins mediate eosinophil recruitment to the lung. In vitro studies indicate that α4β1 and αMβ2 are the principal integrins mediating eosinophil adhesion, including to vascular cell adhesion molecule-1 and the novel αMβ2 ligand periostin. In vivo, blood eosinophils have intermediate-activity β1 integrins, as judged by mAb N29, apparently resulting from eosinophil binding of P-selectin on the surface of activated platelets, and have a proportion of their β2 integrins in the intermediate conformation, as judged by mAb KIM-127, apparently due to exposure to low concentrations of interleukin-5 (IL-5). Airway eosinophils recovered by bronchoalveolar lavage (BAL) after segmental antigen challenge have high-activity β1 integrins and high-activity αMβ2 that does not require IL-5. Here we review information on how the activation states of eosinophil β1 and β2 integrins correlate with measurements of eosinophil recruitment and pulmonary function in asthma. Blood eosinophil N29 reactivity is associated with decreased lung function under various circumstances in non-severe asthma and KIM-127 with BAL eosinophil numbers, indicating that intermediate-activity α4β1 and αMβ2 of blood eosinophils are important for eosinophil arrest and consequently for recruitment and aspects of asthma.

Attenuation of Chronic Pulmonary Inflammation in A2B Adenosine Receptor Knockout Mice

Pharmacologic evidence suggests that activation of A(2B) adenosine receptors results in proinflammatory effects relevant to the progression of asthma, a chronic lung disease associated with elevated interstitial adenosine concentrations in the lung. This concept has been challenged by the finding that genetic removal of A(2B) receptors leads to exaggerated responses in models of acute inflammation. Therefore, the goal of our study was to determine the effects of A(2B) receptor gene ablation in the context of ovalbumin-induced chronic pulmonary inflammation. We found that repetitive airway allergen challenge induced a significant increase in adenosine levels in fluid recovered by bronchoalveolar lavage. Genetic ablation of A(2B) receptors significantly attenuated allergen-induced chronic pulmonary inflammation, as evidenced by a reduction in the number of bronchoalveolar lavage eosinophils and in peribronchial eosinophilic infiltration. The most striking difference in the pulmonary inflammation induced in A(2B) receptor knockout (A(2B)KO) and wild-type mice was the lack of allergen-induced IL-4 release in the airways of A(2B)KO animals, in line with a significant reduction in IL-4 protein and mRNA levels in lung tissue. In addition, attenuation of allergen-induced transforming growth factor-beta release in airways of A(2B)KO mice correlated with reduced airway smooth muscle and goblet cell hyperplasia/hypertrophy. In conclusion, genetic removal of A(2B) adenosine receptors in mice leads to inhibition of allergen-induced chronic pulmonary inflammation and airway remodeling. These findings are in agreement with previous pharmacologic studies suggesting a deleterious role for A(2B) receptor signaling in chronic lung inflammation.

High-dose but not low-dose mainstream cigarette smoke suppresses allergic airway inflammation by inhibiting T cell function

Epidemiological studies have identified childhood exposure to environmental tobacco smoke as a significant risk factor for the onset and exacerbation of asthma, but studies of smoking in adults are less conclusive, and mainstream cigarette smoke (MCS) has been reported to both enhance and attenuate allergic airway inflammation in animal models. We sensitized mice to ovalbumin (OVA) and exposed them to MCS in a well-characterized exposure system. Exposure to MCS (600 mg/m(3) total suspended particulates, TSP) for 1 h/day suppresses the allergic airway response, with reductions in eosinophilia, tissue inflammation, goblet cell metaplasia, IL-4 and IL-5 in bronchoalveolar lavage (BAL) fluid, and OVA-specific antibodies. Suppression is associated with a loss of antigen-specific proliferation and cytokine production by T cells. However, exposure to a lower dose of MCS (77 mg/m(3) TSP) had no effect on the number of BAL eosinophils or OVA-specific antibodies. This is the first report to demonstrate, using identical smoking methodologies, that MCS inhibits immune responses in a dose-dependent manner and may explain the observation that, although smoking provokes a systemic inflammatory response, it also inhibits T cell-mediated responses involved in a number of diseases.

Desquamative Interstitial Pneumonia with a Remarkable Increase in the Number of BAL Eosinophils

A 57-year old man with desquamative interstitial pneumonia (DIP) showed a marked increase in eosinophils in the bronchoalveolar lavage (BAL) fluid. The patient was referred to our hospital for abnormal shadows on his chest X-ray with no symptoms in May 2007. Computed tomography (CT) showed patchy, peripheral predominate ground-glass opacity. The BAL fluid revealed an increase of the total number of cells, including markedly elevated levels of eosinophils (62.1%), in contrast with only a slight increase of peripheral blood eosinophils, or minimal eosinophils in the alveolar spaces and interstitium of the thoracoscopic lung biopsy specimen. Since the specimens showed findings compatible with a DIP pattern, we diagnosed the patient with DIP. Although it is a rare entity, we should therefore consider DIP in the differential diagnosis when we encounter patients with a marked increase in the number of BAL eosinophils.

Relationship Between Eosinophilia and Levels of Chemokines (CCL5 and CCL11) and IL-5 in Bronchoalveolar Lavage Fluid of Patients With Mustard Gas-Induced Pulmonary Fibrosis

Therefore, this study was designed to analyze the bronchoalveolar lavage (BAL) fluid concentrations of IL-5, RANTES (CCL5) and eotaxin (CCL11) and also to examine the relationship between the percentage and absolute number of the BAL eosinophils and these measured chemokines in patients with sulfur mustard (SM) gas-induced pulmonary fibrosis (PF). Fifteen veterans with mustard gas-induced PF and 14 normal veterans as control group. Pulmonary function tests, tests for D(LCO), computed tomography scans of the chest, analyses of BAL fluids for RANTES (CCL5), eotaxin (CCL11), and IL-5 were performed in all cases. Eosinophilic alveolitis was the predominant feature (p < 0.0001). There were significant differences in CCL5, CCL11, and IL-5 levels of BAL fluid between patients with PF and controls (p < 0.0001, p < 0.0001, and p = 0.001, respectively). The concentrations of CCL5 and CCL11 showed positive correlations with percentage (r = 0.57 and p = 0.03; r = 0.52 and p = 0.04, respectively) and absolute counts (r = 0.54 and p = 0.04, r = 0.53 and p = 0.04, respectively) of BAL eosinophils. There were significant positive correlations between the concentrations of IL-5 and the proportion and total cell number of eosinophils in BAL (r = 0.67 and p = 0.01; r = 0.59 and p = 0.02, respectively) too. A significant correlation between BAL CCL5, CCL11, and IL-5 levels and eosinophils in patients with pulmonary fibrosis due to SM gas inhalation has been demonstrated, suggesting that these C-C chemokines and IL-5 contribute to the recruitment of eosinophils cells in the lung in these victims.

Anti‐tumor necrosis factor‐α antibody treatment reduces pulmonary inflammation and methacholine hyper‐responsiveness in a murine asthma model induced by house dust

Recent studies documented that sensitization and exposure to cockroach allergens significantly increase children's asthma morbidity as well as severity, especially among inner city children. TNF-alpha has been postulated to be a critical mediator directly contributing to the bronchopulmonary inflammation and airway hyper-responsiveness in asthma. This study investigated whether an anti-TNF-alpha antibody would inhibit pulmonary inflammation and methacholine (Mch) hyper-responsiveness in a mouse model of asthma induced by a house dust extract containing both endotoxin and cockroach allergens. A house dust sample was extracted with phosphate-buffered saline and then used for immunization and two additional pulmonary challenges of BALB/c mice. Mice were treated with an intravenous injection of anti-TNF-alpha antibody or control antibody 1 h before each pulmonary challenge. In a kinetic study, TNF-alpha levels within the bronchoalveolar lavage (BAL) fluid increased quickly peaking at 2 h while BAL levels of IL-4, IL-5, and IL-13 peaked at later time-points. Mch hyper-responsiveness was measured 24 h after the last challenge, and mice were killed 24 h later. TNF inhibition resulted in an augmentation of these Th2 cytokines. However, the allergic pulmonary inflammation was significantly reduced by anti-TNF-alpha antibody treatment as demonstrated by a substantial reduction in the number of BAL eosinophils, lymphocytes, macrophages, and neutrophils compared with rat IgG-treated mice. Mch hyper-responsiveness was also significantly reduced in anti-TNF-alpha antibody-treated mice and the pulmonary histology was also significantly improved. Inhibition of TNF significantly reduced eotaxin levels within the lung, suggesting a potential mechanism for the beneficial effects. These data indicate that anti-TNF-alpha antibody can reduce the inflammation and pathophysiology of asthma in a murine model of asthma induced by a house dust extract.

Stimulation of protease‐activated receptor‐2 inhibits airway eosinophilia, hyperresponsiveness and bronchoconstriction in a murine model of allergic inflammation

1. An emerging body of evidence indicates that PGE(2) has a privileged anti-inflammatory role within the airways. Stimulants of protease-activated receptor-2 (PAR(2)) inhibit airway smooth muscle tone in vitro and in vivo predominantly via cyclooxygenase (COX)-dependent generation of prostaglandin E(2) (PGE(2)). Thus, the current study tested the hypothesis that PAR(2)-induced generation of PGE(2) inhibits the development of allergic airways inflammation and hyperresponsiveness. 2. Bronchoalveolar lavage (BAL) fluid recovered from ovalbumin (OVA)-sensitised and -challenged (allergic) mice contained elevated numbers of eosinophils, which peaked at 48 h postchallenge. Intranasal (i.n.) administration of a PAR(2)-activating peptide (PAR(2)-AP) SLIGRL (25 mg kg(-1), at the time of OVA challenge) caused a 70% reduction in the numbers of BAL eosinophils (compared to the scrambled peptide LSIGRL, 25 mg kg(-1)). 3. Pretreatment of allergic mice with either indomethacin (1 mg kg(-1), dual COX inhibitor) or nimesulide (3 mg kg(-1), COX-2-selective inhibitor) blocked SLIGRL-induced reductions in BAL eosinophils. 4. I.n. SLIGRL, but not LSIGRL, inhibited the development of antigen-induced airways hyperresponsiveness. The inhibitory effect of SLIGRL was blocked by indomethacin. 5. Exposure of isolated tracheal preparations from allergic mice to 100 microM SLIGRL was associated with a 5.0-fold increase in PGE(2) levels (P<0.05, compared to 100 microM LSIGRL). SLIGRL induced similar increases in PGE(2) levels in control mice (OVA-sensitised, saline-challenged). 6. I.n. administration of PGE(2) (0.15 mg kg(-1)) to allergic mice significantly inhibited eosinophilia and airways hyperresponsiveness to methacholine. 7. In anaesthetised, ventilated allergic mice, SLIGRL (5 mg kg(-1), i.v.) inhibited methacholine-induced increases in airways resistance. Consistent with this bronchodilator effect, SLIGRL induced pronounced relaxation responses in isolated tracheal preparations obtained from allergic mice. LSIGRL did not inhibit bronchomotor tone in either of these in vivo or in vitro experiments. 8. In summary, a PAR(2)-AP SLIGRL inhibited the development of airway eosinophilia and hyperresponsiveness in allergic mice through a COX-dependent pathway involving COX-2-mediated generation of the anti-inflammatory mediator PGE(2). SLIGRL also displayed bronchodilator activity in allergic mice. These studies support the concept that PAR(2) exerts predominantly bronchoprotective actions within allergic murine airways.

Time Course of Eosinophilic Recruitment and Pulmonary Toxicity Biomarkers in an Allergic Asthma Model in Brown Norway Rats

Allergic asthma is a pulmonary disease characterized by antigen-induced pulmonary eosinophilia, airway hyperresponsiveness, antigen-specific IgE antibody responses, and broncho-constriction. In attempting to elucidate mechanisms associated with the pathogenesis of this disease, a number of animal models have been developed. The current studies were undertaken to develop a model of allergic asthma model in Brown Norway rats. Unlike the neutrophilic inflammatory response to inhaled particles in most strains of rats, inhalation of antigens in sensitized Brown Norway rats results in a complex cellular response which is characterized by a variety of inflammatory cell types, and is dependent on the time course of inflammatory cell recruitment. In characterizing this ovalbumin-challenge model of allergic asthma, it was important to assess the time course of pulmonary inflammation, cell proliferation, and apoptosis. Male Brown Norway rats were sensitized and boosted with intraperitoneal injections of ovalbumin in aluminum hydroxide on experimental days 1 and 8. On days 15-17, rats were challenged by an inhalation exposure to 5% ovalbumin and were evaluated by bronchoalveolar lavage (BAL) at 24 or 48 h postexposure (PE). Control rats were similarly treated to ovalbumin aerosol exposures; however, these animals had been sensitized and boosted with aluminum hydroxide (minus the ovalbumin). Cell differential evaluations demonstrated that the rats exposed for 3 days/24 h postexposure and for 2 days/ 48 h postexposure produced the greatest numbers of BAL eosinophils and corresponding indicators of pulmonary toxicity. It was interesting to note that earlier exposure time periods (i.e., 1 day/24 h PE) generated a predominantly neutrophilic inflammatory response, while longer exposure/postexposure time periods (i.e., 3 days/48 h) produced a predominant mononuclear inflammatory response. Subsequent studies demonstrated that the 2-day/ 48-h protocol produced the optimum eosinophilic, cytotoxic, cell proliferative, and apoptotic response. Histopathological evaluations demonstrated a chronically active alveolitis and bronchiolitis, characterized by epithelial cell proliferation in the airways and inflammatory cell proliferation in the alveoli. Studies are ongoing to assess the cell types undergoing apoptosis in both the airway and parenchymal regions to fully characterize this model in order to assess its relevance and utility for studying asthma in humans.

Differential regulation of cytokine expression after allergen exposure of sensitized rats by cyclosporin A and corticosteroids: Relationship to bronchial hyperresponsiveness

Background: Cyclosporin A and dexamethasone exhibit different effects on allergic airway eosinophilia and bronchial hyperresponsiveness (BHR). Objective: We determined whether these were related to alteration of cytokine expression, particularly with regard to the profile of Th1- versus Th2-derived cytokines. Methods: Brown-Norway rats sensitized with ovalbumin were administered cyclosporine or dexamethasone before ovalbumin aerosol challenge. Bronchial responsiveness was measured 18 to 24 hours after aerosol exposure. Airway cellular influx was determined by bronchoalveolar lavage and tissue immunohistochemistry. The expression of Th1 and Th2 cytokine messenger RNA (mRNA) was analyzed by reverse transcriptase–PCR. Results: Ovalbumin exposure induced significant BHR, with increases in eosinophils, lymphocytes, and neutrophils in bronchoalveolar lavage fluid and an increase in eosinophils, CD2 + and CD8 +, but not CD4 + T cells, in the airway submucosa. IL-2, IFN-γ, IL-4, and IL-5 mRNA expression in the lungs of sensitized ovalbumin-exposed rats was increased ( P < .05) compared with controls. Cyclosporin A had no significant effect on BHR and neutrophil accumulation but reduced the number of bronchoalveolar lavage eosinophils ( P < .002), airway submucosal eosinophils, and CD4 + and CD8 + T cells ( P < .02). It also suppressed the induced mRNA expression of IL-2, IL-4, IL-5, and IFN-γ. By contrast, the inflammatory cell influx and mRNA expression for IL-2, IL-4, and IL-5, as well as BHR, were suppressed by dexamethasone. However, an increase in IL-10 and IFN-γ mRNA expression was found. Conclusion: The differential activities of cyclosporin A and dexamethasone on inflammatory cell influx, particularly neutrophils, or cytokine expression such as IL-10 and IFN-γ may underlie their contrasting effects on BHR. (J Allergy Clin Immunol 1999;104:644-52.)

Modulation of airway hyperresponsiveness and eosinophilia by selective histamine and 5-HT receptor antagonists in a mouse model of allergic asthma.

1. Since both histamine and 5-hydroxytryptamine (5-HT) can be released by murine mast cells, we investigated the possible role of these autacoids on airway hyperresponsiveness (AHR), eosinophil infiltration and serum-IgE levels in a murine model of allergic asthma. 2. Ovalbumin-sensitized mice were exposed to either ovalbumin (2 mg ml(-1)) or saline aerosols on 8 consecutive days. Starting one day before the challenge, animals were injected i.p. twice a day with a 5-HT-type 1 (5-HT1) or type 2 (5-HT2) receptor antagonist (methiotepine, 1.25 or 2.0 mg kg(-1) and ketanserin, 12 mg kg(-1), respectively) or a histamine-type 1 (H1) or type 2 (H2) receptor antagonist (mepyramine, 12 or 20 mg kg(-1) and cimetidine, 10 or 25 mg kg(-1), respectively). Furthermore, animals were injected with a combination of cimetidine and ketanserin or with an alpha-adrenoceptor antagonist (phentolamine, 5 mg kg(-1)). 3. In vehicle-treated ovalbumin-challenged animals airway responsiveness to intravenous injections of methacholine in vivo was significantly (9 fold increase, P<0.01) increased when compared to vehicle-treated saline-challenged animals. Furthermore, ovalbumin challenge of vehicle-treated animals induced a significant increase in both eosinophil numbers in bronchoalveolar lavage (BAL) fluid (0+/-0, vehicle/saline and 15.0+/-5.9 x 10(4) cells vehicle/ovalbumin, P<0.05) and ovalbumin-specific IgE levels in serum (157+/-69 and 617+/-171 units ml(-1), respectively, P<0.05) compared to saline-challenged mice. Virtually no eosinophils could be detected in saline-challenged animals after all different treatments. 4. Treatment with ketanserin or cimetidine resulted in a partial but significant decrease of the ovalbumin-induced AHR compared to ovalbumin-challenged controls (P<0.05) and reduced eosinophil infiltration after ovalbumin challenge by 60% and 58%, respectively. The combination of cimetidine and ketanserin almost completely abolished AHR whereas eosinophilia was decreased by 49%. No effects of these antagonists were observed on IL-16 levels in BAL fluid or on serum antigen-specific IgE levels. Treatment with either the H1-receptor, the 5-HT1-receptor or the alpha-adrenoceptor antagonist, did not decrease the observed ovalbumin-induced airway responsiveness or eosinophilia in vehicle-treated animals. Higher doses of either methiotepine (2.0 mg kg(-1)) or mepyramine (20 mg kg(-1)) did decrease ovalbumin-induced eosinophil infiltration (by 67%, P<0.05 and 73%, respectively), whereas no effects of these antagonists were observed on ovalbumin-specific IgE levels in serum. 5. From these data it can be concluded that both histamine and 5-HT play a role in antigen-induced AHR and eosinophilia in the mouse.

Cellular and mediator responses twenty-four hours after local endobronchial allergen challenge of asthmatic airways

The effects of acute allergen exposure on bronchoalveolar lavage cells and mediators and mucosal inflammatory cells were evaluated in 10 subjects with atopic asthma who underwent lavage and biopsy 24 hours after segmental endobronchial allergen challenge. Increased numbers of bronchoalveolar lavage eosinophils were retrieved from the allergen-challenged sites compared with the saline-challenged sites (mean 21.4 vs 1.5 × 10 3 cells/ml; p < 0.02). Numbers of neutrophils and proportions of CD4 +, CD8 +, CD25 +, and HLA-DR + T cells were similar at the saline- and allergen-challenged sites. In contrast to the bronchoalveolar lavage findings, eosinophil numbers were not increased in the bronchial submucosa or epithelium. There was also no significant difference in neutrophils, mast cells, CD3 +, CD4 +, or CD8 + T cells in the submucosa after allergen challenge, but the number of activated (CD25 +) T lymphocytes in the mucosa did increase after allergen challenge. Allergen challenge did not induce any significant change in endothelial expression of P-selectin, E-selectin, intercellular adhesion molecule-1, or vascular cell adhesion molecule-1. CD11a + and very late antigen-4 + cell numbers were similar in the saline- and allergen-challenged sites. This study suggests that in patients with very mild asthma, local allergen challenge induces persistent bronchoalveolar lavage eosinophilia, but the recruitment process seems to have diminished or ceased by 24 hours. (J A LLERGY C LIN I MMUNOL 1996;98:133-43.)

Eosinophil infiltration precedes development of airway hyperreactivity and mucosal exudation after intranasal administration of interleukin-5 to mice

Recently, we demonstrated that antibody to interleukin-5 (IL-5) prevents the infiltration of eosinophils in the respiratory airways and the development of bronchial hyperreactivity in an animal model of allergic asthma. In this study we investigated the influence of long-term intranasal administration of IL-5 on airway responsiveness in vitro, the infiltration of inflammatory leukocytes, and mucosal exudation. Mice (BALB/c) received 1 μg of recombinant human IL-5 in 30 μl of saline solution or vehicle alone twice a day for 1, 3, and 7 days. At 3 and 7 days after IL-5 administration, the number of bronchoalveolar lavage eosinophils increased approximately fourfold and sixfold, respectively. Blood eosinophil numbers showed a similar increase. In addition, 7 days after IL-5 treatment, total lung eosinophil peroxidase activity was significantly increased by 170% as compared with controls. The maximal responsiveness of the trachea in vitro to methacholine was significantly increased by 34%, as compared with controls, only at 7 days after IL-5 administration. Furthermore, mucosal exudation was also only increased significantly at 7 days after IL-5 administration. It can be concluded that the IL-5–induced eosinophil infiltration precedes the development of airway hyperreactivity and mucosal exudation. ( J Allergy Clin Immunol 1995;96:104-12.)

Adoptive transfer of allergic airway responses with sensitized lymphocytes in BN rats.

To evaluate the role of lymphocytes in the pathogenesis of allergic bronchoconstriction, we investigated whether allergic airway responses are adoptively transferred by antigen-primed lymphocytes in Brown Norway (BN) rats. Animals were actively sensitized to ovalbumin (OA) or sham sensitized, and 14 d later mononuclear cells (MNCs) were isolated from intrathoracic lymph nodes, passed through a nylon wool column, and transferred to naive syngeneic rats. Recipients were challenged with aerosolized OA or bovine serum albumin (BSA) (5% wt/vol) and analyzed for changes in lung resistance (RL), airway responsiveness to inhaled methacholine (MCh), and bronchoalveolar lavage (BAL) cells. Recipients of MNCs from sensitized rats responded to OA inhalation and exhibited sustained increases in RL throughout the 8-h observation period, but without usual early airway responses. Recipients of sham-sensitized MNCs or BSA-challenged recipients failed to respond to antigen challenge. At 32 h after OA exposure, airway responsiveness to MCh was increased in four of seven rats that had received sensitized MNCs (p = 0.035). BAL eosinophils increased at 32 h in the recipients of both sensitized and sham-sensitized MNCs. However, eosinophil numbers in BAL were inversely correlated with airway responsiveness in the recipients of sensitized MNCs (r = -0.788, p = 0.036). OA-specific immunoglobulin E (IgE) was undetectable by enzyme-linked immunosorbent assay (ELISA) or passive cutaneous anaphylaxis (PCA) in recipient rats following adoptive transfer. In conclusion, allergic late airway responses (LAR) and cholinergic airway hyperresponsiveness, but not antigen-specific IgE and early responses, were adoptively transferred by antigen-primed lymphocytes in BN rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of rolipram on responses to acute and chronic antigen exposure in monkeys.

The following study was performed to test the hypothesis that treatment with rolipram, a specific inhibitor of phosphodiesterase (PDE) IV, should inhibit many pulmonary responses to acute and chronic antigen challenge in atopic monkeys by elevating intracellular cAMP and subsequently inhibiting leukocyte function. Monkeys received subcutaneous injections of either vehicle (2% DMSO) or 10 mg/kg of rolipram 1 h before exposure to Ascaris suum antigen (Ag). Acute responses to Ag, including bronchoconstriction, pulmonary leukocyte infiltration, and cytokine production, were monitored before and 4 h after single Ag aerosol administration. To monitor the effects of rolipram on chronic Ag exposure, a 10-d, multiple-Ag protocol, previously demonstrated to induce airway hyperresponsiveness (AHR) to methacholine (MCh), was performed. Ag exposure increased respiratory system resistance (Rrs) 221.7 +/- 31.88% (n = 5). This increase in Rrs was not significantly altered by rolipram. Rolipram significantly (p < 0.002) increased cAMP levels in bronchoalveolar lavage (BAL) leukocytes 1 h after administration (n = 5). Ag-induced increases in BAL IL-8 and TNF were significantly reduced by rolipram, but IL-1 beta and IL-6 increases were unaffected (n = 9). Ag-induced increases in BAL eosinophils and neutrophils were significantly reduced by rolipram (n = 9). In the multiple-Ag protocol (n = 7), rolipram significantly reduced both the number of BAL eosinophils (p < 0.02) and the development of AHR (p < 0.002). Despite its inability to inhibit acute Ag-induced bronchoconstriction, rolipram was protective against acute and chronic inflammatory responses to Ag and prevented the development of AHR, suggesting that selective PDE-IV inhibition is a relevant target for asthma therapy.