Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs

Background Endogenous adenosine levels are raised in the lungs during asthma attacks. 5′‐adenosine monophosphate (5′‐AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea‐pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper‐reactivity (AHR) and inflammatory cell recruitment to the lungs. Objective The aim of this study was to investigate the roles of A₁, A_2A, A_2B and A₃ adenosine receptors in these responses to inhaled 5′‐AMP in sensitized guinea‐pigs. Comparisons were made with the effect of dexamethasone treatment on 5′‐AMP‐induced responses. Methods Functional airways responses to inhaled 5′‐AMP (3 and 300 mm ) of actively sensitized, conscious guinea‐pigs were determined by whole‐body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mm ) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. Results 5′‐AMP at 3 mm caused an immediate bronchoconstriction (EAR), whereas 300 mm caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A_2A receptor antagonist, ZM241385, further enhanced cell influx after 5′‐AMP inhalation (3 and 300 mm ), and blocked the immediate bronchodilator response to 300 mm 5′‐AMP, exposing an EAR. The A_2B receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5′‐AMP (300 mm ). The A₃ receptor antagonist, MRS1220, inhibited 5′‐AMP‐induced inflammatory cell influx. The A₁ receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5′‐AMP inhalation (300 mm ). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5′‐AMP (300 mm ). Conclusion All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5′‐AMP in sensitized guinea‐pigs.     

Early- and late-phase bronchoconstriction, airway hyper-reactivity and cell influx into the lungs, after 5'-adenosine monophosphate inhalation: comparison with ovalbumin

Background Antigen inhalation in atopic asthmatic patients results in an early asthmatic response (EAR), accompanied by a late asthmatic response (LAR) in 60% of patients. Inhaled 5′‐adenosine monophosphate (5′‐AMP) causes immediate bronchoconstriction in asthmatics but not in normal subjects. Objectives The aims of this study were to investigate whether 5′‐AMP can produce a LAR, airway hyper‐reactivity (AHR) and cell influx to the lungs, in a sensitized guinea‐pig model of asthma, and to compare with the profile of activity after ovalbumin (OVA) inhalation. Methods Airway responses to inhaled OVA (10 μg/mL) and 5′‐AMP (3 and 300 mm ) of actively sensitized, conscious guinea‐pigs were determined by whole body plethysmography as the change in specific airway conductance (sG_aw). Inhaled histamine (1 mm ) was used to investigate AHR, and cell influx was determined by bronchoalveolar lavage (BAL). Results Exposure to OVA revealed an EAR, and LAR at 6 h post‐challenge. AHR to histamine occurred 24 h after challenge together with a significant increase in total and differential (eosinophils and macrophages) cell counts. Low dose 5′‐AMP (3 mm ) produced an EAR, LAR at 6 h after challenge, and AHR to histamine 12 h post‐challenge. No AHR occurred 24 h after inhalation. Total and macrophage cell counts were increased significantly 6, 12 and 24 h after exposure. Bronchodilatation followed high dose 5′‐AMP (300 mm ), followed by a LAR at 6 h. AHR to histamine occurred 12 h after challenge, but not at 24 h. A significant increase in total and differential (eosinophils and macrophages) cell counts occurred 6, 12 and 24 h post‐exposure. No changes were observed in non‐sensitized guinea‐pigs. Conclusion OVA challenge revealed an EAR, LAR, cell influx and AHR in a guinea‐pig model of asthma. This study demonstrated for the first time that a LAR and AHR to histamine can be revealed following 5′‐AMP inhalation, in sensitized but not unsensitized guinea‐pigs. Cell influx at 6, 12 and 24 h post‐challenge suggests that it may be associated with the LAR and AHR.     

Provocation with adenosine 5''-monophosphate, but not methacholine, induces sputum eosinophilia

INTRODUCTION: Bronchial hyper-responsiveness is usually measured with direct stimuli such as methacholine (MCh) or histamine. Adenosine 5'-monophosphate (AMP), which acts indirectly via the secondary release of mediators, is another stimulus to measure bronchial hyper-responsiveness. AIM: To investigate whether provocation with inhaled AMP itself initiates an inflammatory response resulting in an influx of eosinophils into the airway lumen. METHODS: We have included 21 non-smoking atopic asthmatic subjects (mean FEV1 101% predicted, mean age 34 years). Each subject performed three sputum inductions on different days, at least seven days apart: one without previous provocation, one hour after PC20 methacholine, and one hour after PC20 AMP. RESULTS: After provocation with AMP, but not methacholine, the percentage of sputum eosinophils increased significantly (from 1.9+/-0.5% to 4.5+/-1% (P<0.01) and 1.9+/-0.5% (P=0.89)). No changes in the percentages of neutrophils, lymphocytes, macrophages, or bronchial epithelial cells were found. CONCLUSION: A provocation test with AMP leads to an increased percentage of sputum eosinophils. This observation cannot be explained by a non-specific response of the airways to a vigorous bronchoconstriction, since methacholine had no effect on inflammatory cells.     

Cigarette smoke-induced changes in guinea-pig airway responsiveness to histamine and citric acid

The effect of 50 min cigarette smoke exposure on airway responsiveness to the bronchoconstrictor and tussive effects of histamine and citric acid has been examined in guinea-pigs. Intravenous histamine increased intratracheal pressure (ITP) in anaesthetized guinea-pigs and the dose-response curve was significantly (P less than 0.05) steeper in cigarette smoke- than in air-exposed animals. ED50 values were 11.4 nmol kg-1 (7.4-16.8, 95% confidence interval) and 42.5 nmol kg-1 (28.8-61.4, 95% confidence interval), respectively (P less than 0.05) in smoke- and air-exposed guinea-pigs indicating an enhanced reactivity. However, the sensitivity to intravenous histamine was not changed by the cigarette smoke exposure, and the maximum increase in intratracheal pressure was the same as in control animals (air: 247 +/- 21%, n = 4; smoke: 223 +/- 18%, n = 7). The cigarette smoke-induced hyperresponsiveness to intravenous histamine was not altered by pretreatment with nebulized lidocaine (0.20 M), ipratropium bromide (0.30 mM) or cromoglycate (0.06 M), suggesting that a neural reflex is unlikely to be involved in the development of hyperresponsiveness. Conscious, smoke-exposed guinea-pigs had a significantly (P less than 0.001) reduced responsiveness to citric acid (0.40 M) and cigarette smoke. Both cough and bronchoconstriction were suppressed for about 1 h, but unchanged 24 h after exposure. The hyporesponsiveness to citric acid was inhibited by atropine (1.4 mumol kg-1 i.p.) and may therefore, at least in part, be due to increased airway secretions. The present data demonstrate that inhalation of cigarette smoke may alter guinea-pig airway responsiveness to tussive and bronchoconstrictor stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early and late bronchoconstrictions, airway hyper-reactivity, leucocyte influx and lung histamine and nitric oxide after inhaled antigen: effects of dexamethasone and rolipram

BACKGROUND: Guinea-pig models can provide the essential features of asthma, including early- (EAR) and late- (LAR) phase asthmatic responses, airway hyper-reactivity (AHR) and inflammatory cell influx; however, these components are rarely demonstrated all in the same model. OBJECTIVES: The aim of this study was to establish a conscious guinea-pig model with these essential features of asthma and to correlate these with bronchoalveolar lavage fluid (BALF) histamine and nitric oxide (NO) levels. The model would be validated from the susceptibility of these parameters to standard anti-asthmatic agents, the steroid, dexamethasone, and a phosphodiesterase-4 (PDE4) inhibitor, rolipram. METHODS: Guinea-pigs were sensitized with ovalbumen (OA) (10 microg plus Al2(OH)3 100 mg, intraperitoneal (i.p.)) and 14 days later received inhaled OA (100 microg/mL) or vehicle for 1 h. Airway function was measured by whole-body plethysmography as specific airway conductance (sGaw). Reactivity to inhaled histamine (nose-only, 1 mm, 20 s) was recorded 24 h before and at 6, 12 or 24 h after OA challenge. BALF was obtained to determine the total and differential cell counts, NO and histamine. RESULTS: Guinea-pigs challenged with OA showed an EAR as a fall in (sGaw) (-54.9+/-10.8%), which resolved by 6 h and was followed by an LAR between 7 and 11 h (-30.2+/-8.8%). No bronchoconstriction to inhaled histamine occurred before OA challenge but at 6, 12 or 24 h afterwards, sGaw fell significantly, indicating AHR. At 1 h after OA, macrophages, eosinophils and neutrophils significantly increased in BALF. Macrophages and eosinophils increased further up to 24 h (3- and 44-fold), but neutrophils declined to control levels. BALF histamine levels increased at 0.25 h after OA challenge and peaked at 6 h. BALF NO levels initially fell (44%) 1 h after OA exposure and then progressively rose above control levels. Dexamethasone (20 mg/kg, i.p.) and rolipram (1 mg/kg, i.p.) administered 24 and 0.5 h before and 6 h after OA challenge inhibited leucocyte influx, AHR and the early deficiency and later excess of NO. Dexamethasone but not rolipram attenuated the LAR. CONCLUSIONS: This model displays many of the features of human asthma with predictable responses to dexamethasone and evidence of anti-asthmatic activity by the PDE4 inhibitor, rolipram.     

How does exercise cause asthma attacks?

PURPOSE OF REVIEW: To remind readers that evaporative water loss from the airway surface is the stimulus for exercise-induced bronchoconstriction. To emphasize that recruitment of the peripheral airways determines severity of exercise-induced bronchoconstriction. To draw attention to the potential for injury of the epithelium and for plasma exudation to contribute to the pathogenesis of exercise-induced bronchoconstriction in athletes. To emphasize that many inflammatory mediators are involved in exercise-induced bronchoconstriction and that some are found in both asthmatic and healthy subjects. RECENT FINDINGS: That inflammatory mediators are released into the airways in response to exercise and can be measured by inducing sputum (histamine, cysteinyl leukotrienes) or collecting condensate from exhaled air (cysteinyl leukotrienes and adenosine). The concentration of mediators was reduced in response to a combination of loratadine and montelukast. Exercise is a stimulus for upregulating the genes coding for the 5-lipoxygenase pathway in healthy subjects. SUMMARY: Dehydration of the airways results in release of mediators. The likely source of these mediators is the mast cell. Epithelial injury occurs in exercise-induced bronchoconstriction. The process of repair may contribute to the development of airway hyperresponsiveness in healthy subjects. Measuring the airway response to exercise, or a surrogate for exercise, as an indicator of airway hyperresponsiveness is warranted in patients with symptoms of asthma.     

Effect of aerosolized administration of KF19514, a phosphodiesterase 4 inhibitor, on bronchial hyperresponsiveness and airway inflammation induced by antigen inhalation in guinea-pigs

BACKGROUND: Although phosphodiesterase (PDE) 3 and 4 inhibitors have received much attention for the treatment of bronchial asthma, systemic adverse effects have also been reported. OBJECTIVE: The purpose of this study was to investigate the effect of inhaled olprinone, a newly developed PDE3 inhibitor, and KF19514, a PDE1 and 4 inhibitor, on antigen-induced airway reactions in guinea-pigs. METHODS: Fifteen minutes after inhalation of olprinone (0.1 or 1.0 mg/mL) and KF19514 (0.1 or 0.01 mg/mL), animals were given an antigen challenge. Bronchial hyper-responsiveness and bronchoalveolar lavage fluid cell analysis were performed 24 h after the antigen challenge. RESULTS: Inhalation of olprinone and KF19514 caused a dose-related inhibition of antigen-induced bronchoconstriction. Antigen inhalation significantly increased bronchoconstrictor responses to methacholine, and airway accumulation of neutrophils and eosinophils, 24 h after the antigen challenge. These responses were dose-dependently prevented by KF19514, but not by olprinone. CONCLUSION: The results indicate that inhaled PDE inhibitors might be useful for treatment of bronchial asthma.     

Antibodies directed against nerve growth factor inhibit the acute bronchoconstriction due to allergen challenge in guinea‐pigs

BACKGROUND: We have previously demonstrated that the administration of nerve growth factor (NGF) to guinea-pigs results in airway hyper-responsiveness within 1 h. OBJECTIVE: In the present study we document the involvement of NGF in the acute allergic airway response. METHODS: Guinea-pigs that are sensitized to ovalbumin show an acute bronchoconstriction directly after challenge with ovalbumin. RESULTS: Intratracheal application of 10 microg of antibodies directed against NGF (anti-NGF) 1 h before the challenge reduces the acute severe bronchoconstriction to approximately 40% and the sustained bronchoconstriction to approximately 20% of the reaction in controls. This shows a high potency of anti-NGF in diminishing the direct bronchoconstriction. Inhibition of the tyrosine kinases of the tyrosine kinase receptor A, the high-affinity receptor for NGF, has no effect on the bronchoconstriction. Therefore, we postulate that the p75, the low-affinity receptor for neurotrophins, is responsible for the acute bronchoconstriction. Our findings suggest a role for NGF in the induction of the acute asthmatic reaction. CONCLUSION: These findings offer a new potential therapeutic strategy for the treatment of allergic asthma.     

Early and late phase bronchoconstrictions in conscious sensitized guinea-pigs after macro- and microshock inhalation of allergen and associated airway accumulation of leukocytes

Guinea-pigs were sensitized by i.p. injection of 10 μg OA and 100 mg aluminium hydroxide in 1 ml normal saline. Fourteen to twenty-one days after sensitization, animals were exposed to macroshock (1% OA for 2 min) or microshock (0.01% for 60 min) inhalation challenges with OA. Animals were protected against fatal anaphylaxis in the case of macroshocks with mepyramine (30mg/kg i.p.) 30 min before exposure. Specific airway conductance (sG_aw) was measured in conscious animals by whole body plethysmography at intervals up to 72h after challenge. An early phase bronchoconstriction peaked significantly (P < 0.05) at 15 min after both macroshock and microshock OA exposures, with maximum falls in sG_aw of 70.8 ± 3.8 and −40.0 ± 5.9%, respectively. These had resolved after 5 h. A late phase bronchoconstriction peaked variably between 17 and 24 h: the mean peak falls in sG_aw after the macro- and microshock challenges were significantly different from baseline (P < 0.05), at −21.6 ± 3.7 and −38.0 ± 3.9%, respectively. Control exposures of OA-sensitized guinea-pigs to saline for either 2 or 60 min, in place of OA, produced no significant variation in sG_aw values over the predicted early and late phases. Bronchoalveolar lavage (BAL) performed at 5 or 24h after OA challenge revealed significant increases in total cell numbers (P < 0.05) at 5 and 24 h after the OA macroshock challenge and at 24 h after the microshock, compared with saline challenges. Differential cell counts showed a significant (P < 0.05) increase in the proportion of neutrophils at 5 h and of neutrophils and eosinophils at 24 h after the macroshock exposure to OA, compared with saline controls. A significant (P < 0.05) increase in the proportion of eosinophils also occurred in BAL fluid at 24 h after microshock OA challenge. Neutrophils, however, did not alter at 24 h, yet a late phase bronchoconstriction was recorded. Thus, macroshock (with mepyramine cover) and microshock (without mepyramine cover) OA challenges result in both early and late phase bronchoconstrictions. The late phase is associated with influx of eosinophils in both models but neutrophils only appear after the macroshock, indicating that late phase responses may not involve neutrophil infiltration to the airways.     

Adenosine,inflammation and asthma – a review

Adenosine is a ubiquitous molecule present in every cell of the human body. It has a wide range of physiological functions mediated predominantly through specific cell surface adenosine receptors. Adenosine has both pro- and anti-inflammatory effects and acts on inflammatory and resident immune cells and antioxidant enzymes. The elevation of adenosine in the bronchoalveolar lavage (BAL) fluid of asthmatics combined with its bronchoconstrictor effect on the airways in asthmatics has led to increased research into the contribution of adenosine in the pathophysiology of inflammation and asthma. This review looks at the airway response to adenosine and at the interaction of adenosine with mast cells and basophils.Received 3 October 2003; returned for revision 2 December; accepted by A. Falus 23 December 2003     

Effects of mediator antagonism on mannitol and adenosine monophosphate challenges

BACKGROUND: Airway hyper-responsiveness (AHR) to indirect stimuli is a useful non-invasive surrogate inflammatory marker in the evaluation of asthma, while histamine and cysteinyl leukotrienes are important inflammatory mediators. OBJECTIVE: To evaluate AHR to indirect bronchoconstrictor stimuli and time taken to recover following single doses of montelukast 10 mg and desloratadine 5 mg in combination, montelukast 10 mg alone and placebo. METHODS: Fifteen mild-to-moderate persistent asthmatics completed a randomized, double-blind, cross-over study. Patients received encapsulated montelukast 10 mg/desloratadine 5 mg combination, montelukast 10 mg alone and placebo, 10-14 h prior to challenge on two separate occasions. The mannitol threshold dose, AMP threshold concentration and recovery times after challenge were measured along with lung function. RESULTS: Compared to placebo, montelukast/desloratadine conferred improvements (P < 0.05) in adenosine monophosphate (AMP) threshold concentration and mannitol threshold dose: a 3.2-fold (95% CI 2.2-4.6) and 2.4-fold (95% CI 1.7-3.3) difference, respectively, while compared to montelukast this amounted to a 2.0-fold (95% CI 1.2-3.4) and 1.5-fold (95% CI 1.1-2.4) improvement, respectively. Montelukast was not significantly different from placebo. Both montelukast/desloratadine and montelukast compared to placebo, shortened recovery following both challenges (P < 0.05): a 27-min (95% CI 17-37) and 29-min (95% CI 20-36) reduction, respectively, for AMP, and a 27-min (95% CI 17-37) and 26-min (95% CI 17-35) reduction, respectively for mannitol. CONCLUSION: The dissociated effects of single doses of montelukast alone but not montelukast/desloratadine combination on AHR and recovery time, highlights the relative roles of histamine in initiating the bronchoconstrictor response and cysteinyl leukotrienes in sustaining it. Similar improvements in AHR and recovery time were observed following both indirect bronchoconstrictor stimuli.     

Nerve growth factor increases airway responses and decreases levels of exhaled nitric oxide during histamine challenge in an in vivo guinea-pig model.

There is a growing body of evidence supporting the idea that nerve growth factor (NGF) may be involved in the development of asthma-associated symptoms, such as airway hyper-responsiveness. Increased levels of NGF have recently been described in serum and in the airways of asthmatics. We have examined whether exhaled nitric oxide (NO) levels might be altered during the increased airway responses upon NGF treatment in guinea-pigs in vivo. Intravenous (i.v.) administration of histamine normally elicits a rapid peak in insufflation pressure (IP) and in exhaled NO, followed by a period of decreased concentrations of exhaled NO. Anaesthetized guinea-pigs were pre-treated intravenously with either saline, 4 or 80 ng x kg(-1) NGF 30 min before i.v. challenge with 16 microg x kg(-1) histamine. At 80 ng x kg(-1) NGF significantly enhanced the airway obstruction caused by histamine, whereas the peak acute increase in exhaled NO was not enhanced. Following the increase, came a rapid drop, an effect enforced in the NGF treated animals. Subsequently, the time to return to 90% of resting exhaled NO was increased, from 12 min in saline-treated animals to 48 min in NGF-treated animals. Our data confirm that NGF can enhance airway responses to histamine. Moreover, our study shows a decrease in exhaled NO following a histamine challenge, an effect enhanced by NGF. A reduced ability to release exhaled NO may be a mechanism for increased airway responses during elevated NGF levels. The interaction between NGF and airway NO formation, and its relation to airway responses, merit further investigation.     

Airway hyper-responsiveness in allergic asthma in guinea-pigs is mediated by nerve growth factor via the induction of substance P: a potential role for trkA

Background: The neurotrophin nerve growth factor (NGF) has been implicated as a mediator in allergic asthma. Direct evidence that inhibition of NGF-induced activation of neurotrophin receptors leads to improvement of airway symptoms is lacking. We therefore studied the effects of inhibitors of NGF signal transduction on the development of airway hyper-responsiveness (AHR) and pulmonary inflammation in a guinea-pig model for allergic asthma. Methods: Airway responsiveness to the contractile agonist histamine was measured in vivo in guinea-pigs that were sensitized and challenged with ovalbumin (OVA). Inflammatory cell influx and NGF levels were determined in bronchoalveolar lavage fluid (BALF). Substance P, a key mediator of inflammation, was measured in lung tissue by radioimmunoassay, while substance P immunoreactive neurons in nodose ganglia were measured by immunohistochemistry. Results: OVA challenge induced an AHR after 24 h in OVA-sensitized guinea-pigs. This coincided with an increase in the amount of NGF in BALF. Simultaneously, an increase in the percentage of substance P immunoreactive neurons in the nodose ganglia and an increase in the amount of substance P in lung tissue were found. We used tyrosine kinase inhibitors to block the signal transduction of the high-affinity NGF receptor, tyrosine kinase A (trkA). Treatment with the tyrosine kinase inhibitors (K252a or tyrphostin AG879) both inhibited the development of AHR, and prevented the increase in substance P in the nodose ganglia and lung tissue completely whereas both inhibitors had no effect on baseline airway resistance. Neither treatment with K252a or tyrphostin AG879 changed the influx of inflammatory cells in the BALF due to allergen challenge. Conclusions: We conclude that substance P plays a role in the induction of AHR in our model for allergic asthma which is most likely mediated by NGF. As both tyrosine kinase inhibitors AG879 and K252a show a similar inhibitory effect on airway function after allergen challenge, although both tyrosine kinase inhibitors exhibit different non-specific inhibitory effects on targets other than trkA tyrosine kinases, it is likely that the induction of substance P derived from sensory nerves is mediated by NGF via its high-affinity receptor trkA.     

Inhaled cornstarch glove powder increases latex-induced airway hyper-sensitivity in guinea-pigs

BACKGROUND: Breathing is one of the most important modes of sensitization to natural rubber latex (NRL) for health-care workers, a group most at risk. Cornstarch powder (CSp) from medical powdered NRL gloves is known to be an allergen carrier, and sensitization to NRL can occur by inhaling airborne particles from such gloves. OBJECTIVE: The aim of this study was to demonstrate, using an experimental model, which CSp may act as an adjuvant in NRL-induced airway hyper-responsiveness. METHODS: Guinea-pigs were exposed to aerosolized NRL-contaminated CSp or to NRL in saline solution for 1 h every day for 2 weeks. The control groups were exposed either to CSp or to saline alone. An additional group of guinea-pigs was exposed to aerosolized ovalbumin (OVA) in saline. Three weeks after the last exposure, specific bronchial challenges were performed. In addition, Specific IgG and IgG1 in sera and thromboxane (Tx) B(2) levels in bronchoalveolar lavage fluid (BALF) were measured. RESULTS: The NRL challenge caused significant bronchospasm in the animals that had been exposed to NRL compared with those in the control groups (P<0.02). Guinea-pigs exposed to OVA also demonstrated a significant bronchospasm after OVA challenge (P<0.001). The guinea-pigs that had inhaled NRL-contaminated CSp had a significantly higher bronchoconstriction level than those that had inhaled NRL alone (P<0.02). Specific IgG and IgG1 were undetectable in sera from all groups, whereas significant amounts of TxB(2) (P<0.001) were found in the lungs of the guinea-pigs exposed to NRL or OVA. CONCLUSION: Inhaling CSp increases the airway response to NRL. The fact that specific IgG and IgG1 were not detected might be the result of an immune response limited to the airways. This finding is supported by a significant increase of TxB(2) level in the BALF of sensitized guinea-pigs.     

Dissociation of airway responsiveness and bronchoalveolar lavage (BAL) cell composition in sensitized guinea–pigs after daily inhalation of ovalbumin

Summary. The association between inflammatory cell influx, cell activation status and change of airway responsiveness to acelylcholine (ACh) after daily inhalation of ovalbumin (OA) in sensitized guinea–pigs was investigated. Starting 3 weeks after sensitization (OA at 50mg/kg s.c. + i.p.) guinea–pigs were exposed daily to 2% OA (10min: undercover of 0.5Smg/kg mepyramine i.p. 15min before OA) for 2 weeks. Concentration–response curves (CRCs) for inhaled ACh were performed 24 h after the last OA–challenge and 24 h after another single OA–inhalation 1 week later. CRCs for inhaled ACh were neither affected 24 h after the last OA challenge (daily for two weeks) nor 24 h after another OA–inhalation one week later. In contrast, bronchoalveolar lavage (BAL) from repeatedly OA– sensitized/–challenged guinea–pigs immediately after the last CRC showed a significant increase of total cell count by about tenfold and increases in eosinophils by about 20–fold, neutrophils by 30–fold, macrophages by about fivefold and lymphocytes by about tenfold ( P < 0.05. multiple Wilcoxon–test). In contrast, markers of cell activation (EPO, MPO) were significantly decreased ( P < 0.05). Methylprednisolone almost completely prevented these changes in increased cell numbers and decreased cell activation (vs OA contr., P < 0.05). The lack of increased airway hyperresponsiveness despite a massive inflammatory cell influx suggests other factors controlling airway responsiveness than inflammation.     

Inhibitory effect of inhaled procaterol on anaphylactic bronchoconstriction and thromboxane A2 production in guinea-pigs

This study was designed to examine whether an inhaled beta 2-agonist, procaterol, inhibits thromboxane A2 (TXA2) production induced by antigen challenge in passively sensitized guinea-pigs in vivo. Antigen-induced bronchoconstriction was markedly inhibited by pre-treatment with procaterol. Inhaled procaterol significantly reduced in a dose-dependent manner the increment in TXB2 concentration in bronchoalveolar lavage fluid obtained 5 min after antigen challenge. Aerosol administration of procaterol significantly inhibited bronchoconstriction induced by inhaled histamine. These results suggest that inhalation of procaterol has an inhibitory effect on antigen-induced TXA2 production as well as a protective effect against bronchoconstriction induced by bronchoactive agents.     

Bronchoprotective effects of atrial natriuretic peptide against propranolol-induced bronchoconstriction after allergic reaction in guinea pigs

OBJECTIVE: To study the effects of intravenous atrial natriuretic peptide (ANP) on antigen-induced bronchoconstriction, propranolol-induced bronchoconstriction (PIB) after antigen challenge, and histamine-induced bronchoconstriction in guinea pigs. METHODS: Allergic bronchoconstriction was evoked by inhalation of ovalbumin (OA) and PIB was caused when 10 mg/mL of propranolol was inhaled 20 min after OA challenge in passively sensitized and artificially ventilated guinea pigs. 25, 50, 100 and 200 microg/mL of histamine were inhaled for 20 s at 5-min intervals in non-sensitized guinea pigs. RESULTS: Pretreatment with ANP in doses of 0.1 and 1.0 nmol/kg injected intravenously 15 min after antigen challenge reduced PIB in a dose-dependent manner, and 5 min before antigen challenge significantly attenuated PIB but not antigen-induced bronchoconstriction. Intravenous ANP significantly reduced bronchial responses to increasing concentrations of inhaled histamine in a dose-dependent manner. CONCLUSION: These results suggest that ANP possesses protective effects against propranolol-induced and histamine-induced bronchoconstriction, albeit by a non-specific mechanism in guinea pig in vivo.     

Urinary inflammatory mediators and inhalation of hypertonic saline in children

BACKGROUND: The inflammatory mechanisms of hypertonic saline-induced bronchoconstriction are not well understood. METHODS: Seventeen asthmatics with (n=11) and without bronchial hyperresponsiveness (BHR) (n=6) and 18 randomly selected nonatopic nonasthmatic controls without BHR were evaluated by urine samples collected before and 1 h after hypertonic saline provocation test. Histamine, 11beta-PGF2alpha, and LTE4 were analysed by enzyme immunoassay (EIA) and eosinophil protein X (EPX) by radioimmunoassay (RIA). RESULTS: The levels of leukotriene E4 (LTE4) increased significantly after the challenge tests, both in the asthmatics (median: 354 pg/mg pre-challenge vs. 628 pg/mg post-challenge; P=0.05) and in the controls (median: 294 pg/mg pre-challenge vs. 460 pg/mg post-challenge; P <0.01). The levels of histamine also increased significantly in the latter (median: 299 micromol/mg pre-challenge vs. 569 micromol/mg post-challenge; P=0.03). However, the levels of 11beta-PGF2alpha and EPX did not change significantly after the challenge tests either in the asthmatics or in the controls. CONCLUSIONS: The inhalation of hypertonic saline increased urinary excretion of LTE4 both in the asthmatics and in the controls. The slight increase of leukotrienes was enough to induce airway obstruction in some of the asthmatics, because of the hyperresponsiveness in their airways.     

Activation of protease-activated receptor-2 reduces airways inflammation in experimental allergic asthma

BACKGROUND: Proteinase-activated receptors (PAR)-2 are members of the family of G-protein-coupled receptors activated by proteases. These receptors are widely expressed in several tissues and in virtually all cells involved in rhinitis and asthma. In particular, proteinases activating PAR-2 may affect airway functions and play a role in human diseases. OBJECTIVE: Assessment of the role of PAR-2 in bronchoconstriction, airway responsiveness and immune response after allergic challenge, in rabbits sensitized to Par j 1, the major allergen of Parietaria judaica pollen. METHODS: Evaluation of antigen challenge in rabbits treated with PAR-2-activating peptide (PAR-2AP) (SLIGRL) or the scrambled peptide LSIGRL or vehicle immediately before allergen exposure measuring airway responsiveness. Characterization of bronchoalveolar lavage (BAL) following histamine challenge and phenotype analysis of cells by flow cytometry and analysis of cytokine production by quantitative PCR. RESULTS: PAR-2AP pre-treatment, but not the scrambled peptide, was able to significantly inhibit bronchoconstriction, airway hyper-responsiveness and to modulate the immune response induced by allergic challenge in sensitized rabbits. The phenotype analysis of the cells recovered from BAL showed an increase in RLA-DR-positive cells while RTLA-positive cells were unchanged. IFN-gamma and IL-2 production were inhibited, with a concomitant increase in IL-10 of about 10-fold over the control values. CONCLUSIONS: In this experimental model, PAR-2 modulates bronchoconstriction interfering with antigen challenge-induced immune response in rabbits sensitized and challenged to Par j 1.     

Comparison between repeated antigen and acetylcholine-induced bronchoconstriction in development of airway wall thickness in a chronic guinea pig model of asthma]

Asthma is associated with a chronic remodeling of the airways, but it remains to be elucidated whether repeated bronchoconstriction (BC) influences any structural attributes of the lungs. To investigate the role of repeated BC on the morphology of the airways, we chronically exposed guinea pigs to an aerosol of acetylcholine (ACH) over a period of 6 months. The guinea pigs were challenged with either ACH or saline daily for 10 days and thereafter were challenged once a week. To compare repeated BC with a chronic model of allergic inflammation, an additional group of animals were sensitized to aerosolized ovalbumin (OA) and subsequently exposed with OA. The airway wall area and basement membrane (BM) thickness were measured by standard morphometric techniques. Both airway wall area and BM thickness were significantly increased in OA exposed guinea pigs compared with both saline control and AHC-exposed guinea pigs. Our results suggest that persistent bronchoconstriction and/or allergic airway inflammation in asthmatics may contribute to the thickness of the airway wall observed in this disorder. However, repeated acute bronchoconstriction events may not contribute to the increase in the airway wall or BM thickness.