Modulation of eosinophil migration from bone marrow to lungs of allergic rats by nitric oxide

Chronic blockade of nitric oxide (NO) synthesis attenuates the eosinophil infiltration into airways of allergic rats. This study was designed to investigate whether the inhibition of eosinophil influx to the lung of allergic rats reflects modifications in the pattern of cell mobilization from the bone marrow to peripheral blood and/or to lung. Male Wistar rats were treated with N(omega)-nitro-l-arginine methyl ester (l-NAME; 20mg/rat per day) for 4 weeks and sensitized with ovalbumin (OVA). In control rats, the pulmonary OVA-challenge promoted an early (24h) increase in the bone marrow eosinophil population that normalized at 48 h after OVA-challenge, at which time the eosinophils disappeared from the blood and reached the lungs in mass. In l-NAME-treated rats, an accumulation of eosinophils in bone marrow was observed at 24 and 48 h post-OVA-challenge. No variation in this cell type number was observed in peripheral blood and bronchoalveolar lavage throughout the time-course studied. In control rats, the adhesion of bone marrow eosinophils to fibronectin-covered wells was significantly increased at 24h after OVA-challenge, whereas in l-NAME-treated rats the increased adhesion was detected at 48 h. A 32% decrease in the expression of inducible nitric oxide synthase (iNOS) (but not endothelial nitric oxide synthase; eNOS) in eosinophils from l-NAME-treated rats was observed. The levels of IgE, IgG(1) and IgG(2a) were not affected by the l-NAME treatment. Our findings suggest that inhibition of NO synthesis upregulates the binding of eosinophils to extracellular matrix proteins such as fibronectin, producing a delayed efflux of eosinophils from bone marrow to peripheral blood and lungs.     

Mechanisms involved in the rat peritoneal leukocyte migration induced by a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds

DMTI-II is a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds that causes rat inflammatory edema by mechanisms involving activation of mast cells and sensory C-fibers. The present study aimed to further explore the inflammatory mechanisms involved in DMTI-II-induced inflammation, focusing to the leukocyte migration in vivo. Male Wistar rats (250-280 g) were injected with DMTI-II (1-100 μg/cavity), and at 4-24 h thereafter the leukocyte counts in peritoneal lavage were evaluated. DMTI-II caused dose- and time-dependent accumulation of neutrophils and eosinophils. The peritoneal neutrophil influx initiated at 4 h, achieving maximal responses at 16 h after DMTI-II injection (16- and 22-fold increase, respectively). The DMTI-II-induced eosinophil recruitment was observed as early as 4 h achieving the maximal responses at 16 h (12- and 17-fold increase, respectively). The mononuclear cell number increased at 4 h and 16 h (1.5-fold and 1.6-increase, respectively). Prior treatments with dexamethasone, the cyclooxygenase (COX) inhibitors indomethacin and celecoxib, as well as the PAF receptor antagonist PCA4248 largely reduced the neutrophil and eosinophil accumulation. The selective lypoxygenase inhibitor AA861, the tachykinin NK₁ antagonist SR-140333 and the nitric oxide inhibitor L-NAME reduced only the eosinophil number. The eotaxin levels were significantly higher in DMTI-II-injected rats compared with control animals. In conclusion, DMTI-II causes an early migration of eosinophils and neutrophils by mechanisms involving COX-2- and lipoxygenase-derived metabolites, PAF, substance P and NO. The capacity of DMTI-II to recruit eosinophils at early times is likely to reflect the allergen properties of proteinase inhibitors belonging to Kunitz family.     

Obesity enhances eosinophilic inflammation in a murine model of allergic asthma

Background and purpose:

Obesity is associated with deterioration in asthma outcomes. Although airways eosinophil accumulation is characteristic of lung allergic diseases, little is known about the influence of obesity on the allergic eosinophil trafficking from bone marrow to lung tissues, and recruitment to airways lumen. Here, we have assessed the effects of diet-induced obesity on allergic eosinophilic inflammation in mice, examining eosinophil trafficking from bone marrow to airways, and production of T_H1/T_H2 cytokines.

Experimental approach:

C57BL/6 mice fed for 10 weeks with standard chow or high-fat diet were sensitized and challenged with ovalbumin. At 24–96 h post-ovalbumin challenge, bronchoalveolar lavage (BAL) fluid, lung tissue and bone marrow were examined.

Key results:

The high-fat-fed mice exhibited increased body weight and epididymal fat, glucose intolerance and alterations in lipid profile compared with the lean mice. Obesity markedly elevated serum leptin and lowered adiponectin levels. Ovalbumin challenge in obese mice promoted a markedly higher eosinophil accumulation in bone marrow and connective tissue surrounding the bronchial and bronchiolar segments. Eosinophil number in BAL fluid of obese mice was lower at 24 and 48 h. Levels of interleukin (IL)-5, eotaxin, tumour necrosis factor-α and IL-10 in BAL fluid of obese mice were significantly higher than in lean mice.

Conclusions and implications:

Diet-induced obesity enhanced eosinophil trafficking from bone marrow to lung tissues, and delayed their transit through the airway epithelium into the airway lumen. Consequently, eosinophils remain longer in lung peribronchiolar segments due to overproduction of T_H1/T_H2 cytokines and chemokines.     

Inhibition of pulmonary eosinophilia and airway hyperresponsiveness in allergic mice by rolipram: involvement of endogenously released corticosterone and catecholamines

This study investigates the role of adrenal-derived catecholamines and corticosterone on the inhibition by rolipram, a phosphodiesterase (PDE)-4 inhibitor, of pulmonary eosinophilia and airway hyperresponsiveness (AHR) in allergic mice. The following experimental groups were studied in mice sensitized and challenged with ovalbumin (OVA): normal, adrenalectomized, propranolol (beta-adrenoceptor antagonist) and metyrapone (corticosterone synthesis inhibitor) treated. These interventions were studied both in the absence and in the presence of rolipram. Eosinophil numbers in the bronchoalveolar lavage (BAL) and AHR to methacholine were measured 24 h after OVA challenge. Treatment of sensitized mice with rolipram (0.3 - 10 mg kg(-1), p.o.), inhibited pulmonary eosinophilia and the AHR to methacholine in OVA-challenged mice. Adrenalectomy increased the number of eosinophils in the BAL of OVA-challenged mice but had no effect on AHR to methacholine. Adrenalectomy attenuated both the rolipram-induced inhibition of BAL eosinophilia and AHR to methacholine in OVA challenged mice. Propranolol (10 mg kg(-1), p.o.) had no effect on the inhibition of eosinophilia by rolipram but attenuated the inhibition of AHR to methacholine in OVA challenged mice. On the other hand, metyrapone (10 mg kg(-1), p.o.) attenuated the inhibition of eosinophilia by rolipram but had no effect on the inhibition of AHR to methacholine in OVA challenged mice. Metyrapone-treatment alone increased the number of eosinophils in the BAL of OVA-challenged mice. These results identify an important role for adrenal-derived catecholamines and corticosterone on the inhibition of pulmonary eosinophilia and AHR by rolipram in allergic mice.     

Long-term methylglyoxal intake aggravates murine Th2-mediated airway eosinophil infiltration

Asthma outcomes is aggravated in obese patients. Excess of methylglyoxal (MGO) in obese/diabetic patients has been associated with diverse detrimental effects on cell function. This study aimed to evaluate the effects of long-term oral intake of MGO on ovalbumin-induced eosinophil inflammation. Male C57/Bl6 mice received 0.5% MGO in the drinking water for 12 weeks. Mice were sensitized and challenged with ovalbumin (OVA), and at 48 h thereafter, bronchoalveolar lavage (BAL) fluid and lungs were collected for cell counting, morphological analysis, and ELISA, mRNA expressions and DHE assays. In MGO-treated mice, OVA challenge significantly increased the peribronchiolar infiltrations of inflammatory cells and eosinophils compared with control group. Higher levels of IL-4, IL-5, and eotaxin in BAL fluid were also detected in MGO compared with control group. In addition, lung tissue of MGO-treated mice displayed significant increases in mRNA expressions of NF-κB and iNOS whereas COX-2 expression remained unchanged. The high TNF-α mRNA expression observed in lungs of OVA-challenged control mice was not further increased by MGO treatment. In MGO group, OVA-challenge increased significantly the NOX-2 and NOX-4 mRNA expressions, without affecting the NOX-1 expression. Levels of reactive-oxygen species (ROS) were significantly higher in lungs of MGO-treated mice, and no further increase by OVA-challenge was observed. In conclusion, 12-week intake of MGO exacerbates Th2-mediated airway eosinophil infiltration by activation of NF-kB/iNOS-dependent signaling pathway and positive regulation of NOX-2 and NOX-4 in the lung tissues. Scavengers of MGO could be an option to prevent obesity-related asthma.     

Effects of single or repeated amphetamine treatment and withdrawal on lung allergic inflammation in rats

The effects of single or repeated amphetamine (AMPH) treatment and those of AMPH withdrawals on immune-mediated lung inflammatory response were studied in rats. Two experiments were done. In the first, rats egg-albumin (OVA) sensitized were singularly or repeatedly (21 days, once daily) treated with AMPH (1.0 mg/kg) or with a similar number and volume of 0.9% NaCl. The OVA aerosol challenge was performed 12 h after the single or last repeated AMPH treatment and also 72 and 120 h after AMPH withdrawal. In the second experiment, the effects of reserpine (1.0 mg/kg/day for 5 consecutive days) on single AMPH actions on lung allergic response of rats were analyzed. Single and repeated AMPH treatment induced opposite actions on Bronchoalveolar lavage fluid (BAL) cellularity of allergic rats: single treatment decreased and repeated treatment increased the total number of cells as well as those of macrophages, neutrophils and eosinophils. Our data also showed that single but not repeated AMPH treatment decreased the number of neutrophils, monocytes and lymphocytes in the peripheral blood, and increased the total number of bone marrow cells in rats sensitized and challenged with OVA. Furthermore, it was shown that reserpine treatment precluded the effects of single AMPH treatment on cellular migration to the lung of OVA-sensitized and challenged rats. It was concluded that AMPH effects on lung inflammatory response and cell recruitment to the lung in allergic rats rely at least partially on corticosterone serum levels. The possible involvement of vesicular monoamine transporter type 2 (VMAT2) with these observed effects was discussed.     

Role of the Mac-1 and VLA-4 integrins, and concomitant Th2-cytokine production, in nitric oxide modulated eosinophil migration from bone marrow to lungs in allergic mice

Although numerous studies demonstrate the participation of nitric oxide (NO) in various inflammatory diseases, the precise function of NO in allergic asthma remains unclear. We investigated whether iNOS inhibition could interfere with the kinetics of VLA-4 and Mac-1 expression and adhesion properties of bone marrow and peripheral blood eosinophils of sensitized mice after antigen exposure. Treatment of allergic mice with 1400 W (iNOS inhibitor) increased the adhesion of bone marrow eosinophils to ICAM-1, but not blood eosinophils, at 24h and 48 h after OVA-challenge. Conversely, adhesion of blood eosinophils from 1400 W-treated mice to VCAM-1 diminished at 24h and was almost completely blocked at 48 h. 1400 W did not induce any change in the adhesion of bone marrow eosinophils to VCAM-1, at 24h, but cells collected 48 h after challenge showed significantly lower adherence. Flow cytometry demonstrated that 1400 W resulted in a significantly increased Mac-1 expression on bone marrow eosinophils at 24h, as compared to control mice. However, at 24h, 1400 W significantly decreased Mac-1 and VLA-4 expressions on blood eosinophils. At 48 h, the expressions of both Mac-1 and VLA-4 returned to previous levels. Results show a temporal effect of iNOS upon Mac-1 expression and function, the chief adhesion molecule involved in the eosinophil efflux from the bone marrow at 24h. In contrast, Mac-1 and VLA-4 were involved in eosinophil mobilization from blood to lungs at 48 h after antigen challenge. Data suggest an important role of the Mac-1 and VLA-4 in the iNOS-modulated migration of eosinophils to the lungs of allergic mice.     

Inflammatory mechanisms underlying the rat pulmonary neutrophil influx induced by airway exposure to staphylococcal enterotoxin type A

Association between staphylococcal infection and pathogenesis of upper airways disease has been reported. This study aimed to investigate the mechanisms underlying the rat pulmonary inflammation induced by airway exposure to staphylococcal enterotoxin A (SEA). SEA (0.3-10 ng trachea(-1)) caused dose-dependent neutrophil accumulation in BAL fluid, reaching maximal responses at 4 h (25-fold increase for 3 ng trachea(-1)). Significant accumulation of both lymphocytes and macrophages in BAL fluid was also observed at 4 h (2.1- and 1.9-fold increase, respectively, for 3 ng trachea(-1)). At later times (16 h), neutrophil counts in bone marrow (immature forms) and peripheral blood increased by 63 and 81%, respectively. SEA failed to directly induce chemotaxis and adhesion of isolated neutrophils. Analysis of mRNA expression for iNOS, COX-2 and CINC-2 in lung tissue showed an upregulation of these enzymes, which paralleled elevated levels of LTB4, PGE2, TNF-alpha, IL-6 and NO2- in BAL fluid. Expression of CINC-1 was unchanged, whereas CINC-3 was reduced in SEA-treated rats. Incubation of isolated alveolar macrophages with SEA (3 microg ml(-1)) resulted in significant elevations of TNF-alpha and NO2- levels in the cell supernatants. Dexamethasone (0.5 mg kg(-1)), celecoxib (3 mg kg(-1)) and compound 1400 W (5 mg kg(-1)) markedly reduced SEA-induced lung neutrophil influx and NO2- levels in BAL fluid. The lipoxygenase inhibitor AA-861 (100 microg kg(-1)) partly inhibited the neutrophil influx in SEA-treated rats without modifying the NO2- levels. None of these treatments reduced the number of mononuclear cells in BAL fluid (except of dexamethasone, which abolished the increased lymphocyte counts). Our study shows that airways exposure to SEA results in marked neutrophil influx through mechanisms involving increased expressions of CINC-2, iNOS and COX-2, as well as enhanced production of NO, PGE2, LTB4, TNF-alpha and IL-6.     

Role of adenosine in airway inflammation in an allergic mouse model of asthma

In the present study, we examined dynamic changes in cellular profile of bronchoalveolar lavage (BAL) fluid after adenosine challenge in ragweed sensitized and challenged mice. Mice systemically sensitized and airway challenged with ragweed showed marked airway inflammation manifesting increased eosinophils, lymphocytes, neutrophils and activated macrophages in BAL. Adenosine challenge further enhanced influx of inflammatory cells into BAL, notably neutrophils from 1 to 72 h and eosinophils from 1 to 48 h time-points (p<0.05), which sharply rose at 6-h time-point following adenosine challenge. Greater infiltration of lymphocytes into BAL was observed at 1 and 72 h and macrophages from 6 to 72 h (p<0.05) after adenosine challenge. Accordingly, markers of eosinophils, neutrophils and mast cells were analyzed at 6-h time-point after adenosine challenge. Adenosine challenge significantly increased the levels of eosinophil peroxidase, neutrophil myeloperoxidase and beta-hexosaminidase in BAL. There were more significant effects of adenosine challenge on the degranulation of mast cells in the lung than that in blood. The chemoattractant, eotaxin, was detected in BAL, which increased after adenosine challenge. Theophylline, a non-specific adenosine receptor antagonist, prevented adenosine-enhanced infiltration of inflammatory cells and their respective markers. Our findings suggest that adenosine plays an important role in airway inflammation in an allergic mouse model.     

Effect of a novel interleukin-5 receptor antagonist, YM-90709 (2,3-dimethoxy-6,6-dimethyl-5,6-dihydrobenzo[7,8]indolizino[2,3-b]quinoxaline), on antigen-induced airway inflammation in BN rats

Interleukin-5 (IL-5) plays an important role in the activation of eosinophils in the allergic inflammation in conditions such as asthma, rhinitis, and atopic dermatitis. A newly synthesized compound, YM-90709 (2,3-dimethoxy-6,6-dimethyl-5,6-dihydrobenzo[7,8]indolizino[2,3-b]quinoxaline), was previously reported to inhibit the binding of IL-5 to its receptor (R) on human eosinophils and eosinophilic HL-60 clone 15 cells. However, it did not inhibit the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to its receptor on the same cells. In this study, the intravenous injection of YM-90709 resulted in the inhibition of antigen-induced infiltration of eosinophils and lymphocytes, but not neutrophils or monocytes, into the bronchoalveolar lavage fluid (BALF) of Brown-Norway (BN) rats, with ED50 values of 0.32 mg/kg and 0.12 mg/kg, respectively. Two glucocorticoids, dexamethasone and prednisolone, inhibited neutrophil, eosinophil, and lymphocyte infiltration into the BALF. However, both significantly reduced the number of peripheral blood leukocytes and bone marrow leukocytes. In contrast, YM-90709 did not affect the peripheral blood leukocytes or the bone marrow leukocytes. These results indicate that, in this model, YM-90709, which is a novel IL-5 R antagonist, inhibits antigen-induced eosinophil and lymphocyte recruitment into the airway, without any suppressive effects on peripheral blood leukocytes or bone marrow leukocytes, in contrast to the glucocorticoids.     

EFFECT OF NITROGEN DIOXIDE ON OVALBUMIN-INDUCED ALLERGIC AIRWAY DISEASE IN A MURINE MODEL

The effect of exposure to irritant air pollutants on the development of allergic airway disease is poorly understood. This study examines the effects of the lower respiratory tract irritant, NO 2 , on the outcome of ovalbumin (OVA)-induced allergic airway disease. Male and female C57Bl/6 mice were sensitized by weekly intraperitoneal (ip) OVA injections for 3 wk followed by daily 1-h OVA aerosol inhalation challenge for 3 or 10 d. Initially, mice were exposed daily for 3 d to air or 0.7 or 5 ppm NO 2 for 2 h following each OVA aerosol challenge. OVA exposure resulted in pronounced lower airway inflammation, as evidenced by a significant increase in bronchoalveolar lavage (BAL) total cellularity and eosinophil levels. BAL eosinophil levels were significantly lower in OVA-NO 2 compared to OVA-air animals. The reduction was similar at both NO 2 exposure concentrations. In a subsequent study, sensitized animals were exposed for 3 or 10 d to aerosolized OVA followed by air or 0.7 ppm NO 2 . BAL eosinophils were again reduced at 3 d by OVA-NO 2 exposure compared to OVA-air mice. At 10 d the eosinophilia was virtually abolished. This reduction in OVA-induced cellular inflammation by NO 2 was confirmed by histopathological analysis. Contrary to expectations, exposure to NO 2 during the aerosol challenge to OVA dramatically diminished the outcome of allergic disease in lungs as measured by airway cellular inflammation.     

Differential effects of formaldehyde exposure on the cell influx and vascular permeability in a rat model of allergic lung inflammation

Exposure to air pollutants such as formaldehyde (FA) leads to inflammation, oxidative stress and immune-modulation in the airways and is associated with airway inflammatory disorders such as asthma. The purpose of our study was to investigate the effects of exposure to FA on the allergic lung inflammation. The hypothesized link between reactive oxygen species and the effects of FA was also studied. To do so, male Wistar rats were exposed to FA inhalation (1%, 90 min daily) for 3 days, and subsequently sensitized with ovalbumin (OVA)-alum by subcutaneous route. One week later the rats received another OVA-alum injection by the same route (booster). Two weeks later the rats were challenged with aerosolized OVA. The OVA challenge of rats upon FA exposure induced an elevated release of LTB₄, TXB₂, IL-1β, IL-6 and VEGF in lung cells, increased phagocytosis and lung vascular permeability, whereas the cell recruitment into lung was reduced. FA inhalation induced the oxidative burst and the nitration of proteins in the lung. Vitamins C, E and apocynin reduced the levels of LTB₄ in BAL-cultured cells of the FA and FA/OVA groups, but increased the cell influx into the lung of the FA/OVA rats. In OVA-challenged rats, the exposure to FA was associated to a reduced lung endothelial cells expression of intercellular cell adhesion molecule 1 (ICAM-1). In conclusion, our findings suggest that FA down regulate the cellular migration into the lungs after an allergic challenge and increase the ability of resident lung cells likely macrophages to generate inflammatory mediators, explaining the increased lung vascular permeability. Our data are indicative that the actions of FA involve mechanisms related to endothelium–leukocyte interactions and oxidative stress, as far as the deleterious effects of this air pollutant on airways are concerned.     

Systemic and local dexamethasone treatments prevent allergic eosinophilia in rats via distinct mechanisms.

We have studied the effect of local and systemic treatment with dexamethasone for prevention of the pleural eosinophilia triggered by allergen in actively sensitised Wistar rats. Parallel changes in blood and marrow eosinophil numbers were assessed for comparison. The intrapleural (i.pl.) injection of ovalbumin into ovalbumin-sensitised animals led to a long-lasting pleural fluid eosinophilia which peaked from 24 to 72 h post-challenge. At these time points, there was a significant 2- to 3-fold increase in the blood eosinophil numbers, whereas the bone marrow number of mature eosinophils remained unaltered. Systemic treatment with dexamethasone (0.05-0.5 mg/kg, i.p.) abolished the pleural and blood eosinophilia observed 24 and 48 h post-challenge, also causing a significant reduction in marrow eosinophil numbers. Despite being unable to alter blood and bone marrow eosinophil numbers, the local i.pl. administration of dexamethasone (2.5-10 microg/cavity) inhibited dose dependently the allergen-induced pleural eosinophil influx at 24 h but not at 48 h post-challenge. This treatment also shortened the time course of eosinophil accumulation in the pleural space from the 48 h time point on. We conclude that the effect of systemic but not of local treatment with dexamethasone on allergen-induced eosinophil recruitment is well correlated with the inhibition of eosinophil production in bone marrow. In contrast, low amounts of dexamethasone injected into the pleural space seem to affect locally eosinophil recruitment and survival.     

Effect of nitrogen dioxide on ovalbumin-induced allergic airway disease in a murine model

The effect of exposure to irritant air pollutants on the development of allergic airway disease is poorly understood. This study examines the effects of the lower respiratory tract irritant, NO(2), on the outcome of ovalbumin (OVA)-induced allergic airway disease. Male and female C57Bl/6 mice were sensitized by weekly intraperitoneal (ip) OVA injections for 3 wk followed by daily 1-h OVA aerosol inhalation challenge for 3 or 10 d. Initially, mice were exposed daily for 3 d to air or 0.7 or 5 ppm NO(2) for 2 h following each OVA aerosol challenge. OVA exposure resulted in pronounced lower airway inflammation, as evidenced by a significant increase in bronchoalveolar lavage (BAL) total cellularity and eosinophil levels. BAL eosinophil levels were significantly lower in OVA-NO(2) compared to OVA-air animals. The reduction was similar at both NO(2) exposure concentrations. In a subsequent study, sensitized animals were exposed for 3 or 10 d to aerosolized OVA followed by air or 0.7 ppm NO(2). BAL eosinophils were again reduced at 3 d by OVA-NO(2) exposure compared to OVA-air mice. At 10 d the eosinophilia was virtually abolished. This reduction in OVA-induced cellular inflammation by NO(2) was confirmed by histopathological analysis. Contrary to expectations, exposure to NO(2) during the aerosol challenge to OVA dramatically diminished the outcome of allergic disease in lungs as measured by airway cellular inflammation.     

Ovalbumin Challenge Following Immunization Elicits Recruitment of Eosinophils but not Bronchial Hyperresponsiveness in Guinea-pigs: Time Course and Relationship to Eosinophil Activation Status

Eosinophils are known to be present in the airways of allergic asthmatics, and have been suggested to contribute to the pathophysiological changes accompanying this condition, particularly hyperresponsiveness to airway spasmogens. However, a causal relationship between pulmonary eosinophil accumulation and bronchial hyperresponsiveness in asthma is not proven. In the present study, the time course of pulmonary cell influx was investigated in an immunized guinea-pig model. Eosinophil activation status was also determined together with the bronchial responsiveness to histamine. Guinea-pigs were sensitized [20 μg ovalbumin (OVA) per animal in Al(OH)₃(0.5 ml) ip] and subsequently challenged with aerosolized OVA (100 μg/ml) for 1 h 18–21 days later. At different time points (1 h to 72 h) after OVA challenge, bronchial responses to iv histamine were measured and bronchoalveolar lavage (BAL) was performed to assess pulmonary cell influx. Eosinophil peroxidase (EPO) and total protein levels were measured in BAL fluid supernatants. Exposure of sensitized animals to aerosolized OVA produced a significant increase (P<0.05 vs. sham immunized) in eosinophil infiltration 24 h later which was sustained up to 72 h. Despite this, OVA challenge did not cause either eosinophil activation, as measured by EPO release, or bronchial hyperresponsiveness to histamine at any of the time points examined. These data show that allergen challenge of sensitized guinea-pigs can elicit airway eosinophil accumulation without accompanying airways hyperresponsiveness or eosinophil activation.     

Airway inflammation and adjuvant effect after repeated airborne exposures to di-(2-ethylhexyl)phthalate and ovalbumin in BALB/c mice

BACKGROUND: Epidemiological studies have suggested an association between exposure to phthalate plasticizers, including di-(2-ethylhexyl)phthalate (DEHP), and increased prevalence of asthma, rhinitis or wheezing. Furthermore, studies in mice have demonstrated an adjuvant effect from DEHP after parenteral administration with the model allergen ovalbumin (OVA). OBJECTIVE: Exposures to DEHP were investigated for adjuvant effects and airway inflammation in a mouse inhalation model. METHODS: BALB/cJ mice were exposed to aerosols of 0.022-13 mg/m(3) DEHP and 0.14 mg/m(3) OVA 5 days/week for 2 weeks and thereafter weekly for 12 weeks. Mice exposed to OVA alone or OVA+Al(OH)(3) served as control groups. Finally, all groups were exposed to a nebulized 1% OVA solution on three consecutive days. Serum, bronchoalveolar lavage (BAL) fluid, and draining lymph nodes were collected 24h later. RESULTS: In the OVA+Al(OH)(3) group, significantly increased levels of OVA-specific IgE and IgG1 in serum as well as of eosinophils in BAL fluid were observed. DEHP affected OVA-specific IgG1 production in a concentration-dependent manner, whereas little effect was seen on IgE and IgG2a. Dose-dependent increases in inflammatory cells were observed in BAL fluids, leading to significantly higher lymphocyte, neutrophil and eosinophil numbers in the OVA+13 mg/m(3) DEHP group. Ex vivo cytokine secretion by cultures of draining lymph nodes suggested that DEHP has a mixed Th1/Th2 cytokine profile. CONCLUSION: Airborne DEHP is able to increase serum IgG1 and lung inflammatory cell levels, but only at very high concentrations. Realistic DEHP levels do not have an adjuvant effect or induce allergic lung inflammation in the present mouse model.     

Enhancement of nasal inflammatory and epithelial responses after ozone and allergen coexposure in Brown Norway rats.

Repeated exposures to ozone cause inflammation and mucous cell metaplasia (MCM) in the nasal mucosa of laboratory animals. Similar cellular responses occur in humans during allergic rhinitis. We tested the hypothesis that exposure to ozone will enhance the inflammatory and epithelial responses associated with allergic rhinitis. Ovalbumin (OVA)-sensitized Brown Norway rats were exposed to ozone (0.5 ppm, 8 h/day) for 1 day or 3 consecutive days. Immediately after each ozone exposure, animals were challenged intranasally (IN) with either sterile saline or OVA dissolved in saline (1%, 50 microg/nasal passage). Twenty-four h after the last IN challenge rats were sacrificed; nasal tissues were removed and processed for light microscopic examination and morphometric analysis of numeric densities of inflammatory and epithelial cell populations and volume densities of intraepithelial mucosubstances. A single OVA challenge caused a significant influx of neutrophils and eosinophils into the submucosa of all nasal tissues. Ozone exposure further enhanced the appearance of eosinophils in the maxilloturbinates of OVA-challenged rats but did not increase inflammation in other nasal tissues. After 3 days of ozone/OVA coexposures, the nasal transitional epithelium lining the maxilloturbinates had increased numbers of epithelial cells as well as the appearance of mucus-containing cells in areas normally absent of these secretory cells (i.e., MCM). Multiple challenges with OVA caused increased epithelial mucosubstances in the respiratory epithelium lining the septum without increasing the number of epithelial cells. Multiple exposures to both ozone and OVA caused greater increases in intraepithelial mucosubstances in the septum than those elicited by OVA alone. These results demonstrate that exposure to ozone exacerbates epithelial and inflammatory responses associated with allergen challenge. In addition, coexposure of these agents enhanced the induced production of nasal mucosubstances caused by either agent alone.     

The Effect of Selective Phosphodiesterase Inhibitors in Comparison with other Anti-asthma Drugs on Allergen-induced Eosinophilia in Guinea-pig Airways

Summary: The effects of isoenzyme selective phosphodiesterase (PDE) inhibitors and other anti-asthma drugs on antigen-induced eosinophil recruitment and activation in guinea-pig airways was studied. Guinea-pigs were sensitized and subsequently challenged with aerosolized ovalbumin (OVA). Bronchoalveolar lavage (BAL) was performed 24 h later. A significant increase in eosinophils and eosinophil peroxidase (EPO) was detected in BAL fluid and BAL fluid supernatant respectively from OVA immunized guinea-pigs compared with sham treated animals.Guinea-pigs were treated for 7 days prior to antigen challenge with either the following drugs or the appropriate vehicle (i.p.). The selective β₂ agonist, salbutamol (0.3 mg/kg), the PDE III inhibitor, milrinone (15 mg/kg) and the non-selective PDE inhibitor, trequinsin (1 mg/kg) had no effect on eosinophil number or EPO levels. The PDE IV inhibitor, rolipram (15 mg/kg), the mixed type III/IV PDE inhibitor, benzafentrine (15 mg/kg) and the nonselective PDE inhibitor, aminophylline (31.5 mg/kg) had no effect on eosinophil number but reduced the amount of EPO detected. The anti-inflammatory glucocorticosteroids, beclomethasone (10 mg/kg) and betamethasone (4 mg/kg) and the type IV PDE inhibitor, RP 73401 (5 mg/kg) reduced both eosinophil numbers and EPO levels. These results suggest a role for the type IV PDE isoenzyme in the control of eosinophil recruitment and possibly activation in the airways.     

Acute pulmonary inflammation induced by exposure of the airways to staphylococcal enterotoxin type B in rats

Staphylococcus aureus is a gram-positive bacterium that produces several enterotoxins, which are responsible for most part of pathological conditions associated to staphylococcal infections, including lung inflammation. This study aimed to investigate the underlying inflammatory mechanisms involved in leukocyte recruitment in rats exposed to staphylococcal enterotoxin B (SEB). Rats were anesthetized with pentobarbital sodium and intratracheally injected with either SEB or sterile phosphate-buffered saline (PBS, 0.4 ml). Airways exposition to SEB (7.5-250 ng/trachea) caused a dose- and time-dependent neutrophil accumulation in BAL fluid, the maximal effects of which were observed at 4 h post-SEB exposure (250 ng/trachea). Eosinophils were virtually absent in BAL fluid, whereas mononuclear cell counts increased only at 24 h post-SEB. Significant elevations of granulocytes in bone marrow (mature and immature forms) and peripheral blood have also been detected. In BAL fluid, marked elevations in the levels of lipid mediators (LTB(4) and PGE(2)) and cytokines (TNF-alpha, IL-6 and IL-10) were observed after SEB instillation. The SEB-induced neutrophil accumulation in BAL fluid was reduced by pretreatment with dexamethasone (0.5 mg/kg), the COX-2 inhibitor celecoxib (3 mg/kg), the selective iNOS inhibitor compound 1400 W (5 mg/kg) and the lipoxygenase inhibitor AA-861 (200 microg/kg). In separate experiments carried out with rat isolated peripheral neutrophils, SEB failed to induce neutrophil adhesion to serum-coated plates and chemotaxis. In conclusion, rat airways exposition to SEB causes a neutrophil-dependent lung inflammation at 4 h as result of the release of proinflammatory (NO, PGE(2), LTB(4), TNF-alpha, IL-6) and anti-inflammatory mediators (IL-10).     

Effects of hardwood smoke exposure on allergic airway inflammation in mice

Hardwood smoke (HWS) from wood burning stoves and fireplaces can be a significant contributor to the composition of ambient air pollution. We hypothesize that the inhalation of HWS by ovalbumin (OVA)-sensitized mice with preexisting lung inflammation leads to the exacerbation of allergic airway responses. Two different models were employed to characterize the effects of inhaled wood smoke on allergic airway inflammation. In both models, male BALB/c mice were sensitized by injection with OVA and alum. In one model, mice were challenged by inhalation with OVA 1 day prior to exposure to HWS (30, 100, 300, or 1000 microg particulate matter [PM]/m(3)) for 6 h/day on 3 consecutive days. In the other model, mice were exposed by inhalation to OVA, rested for 11 days, were exposed to HWS for 3 consecutive days, and then were exposed to OVA immediately after the final HWS exposure. Bronchoalveolar lavage (BAL), and blood collection were performed approximately 18 h after the last HWS or OVA exposure. HWS exposure after the final allergen challenge (first model) led to a significant increase in BAL eosinophils only at the 300 microg/m(3) level. In contrast, changes in BAL cells did not reach statistical significance in the second model. There were no HWS-induced changes in BAL interleukin (IL)-2, IL-4, IL-13, and interferon (IFN)gamma levels in either model following OVA challenge. These results suggest that acute HWS exposure can minimally exacerbate some indices of allergic airway inflammation when a final OVA challenge precedes HWS exposure, but does not alter Th1/Th2 cytokine levels.