Repeated antigen challenge induces airway hyperresponsiveness with tissue eosinophilia in guinea pigs

To test the hypothesis that the development of airway hyperresponsiveness (AHR) lasting greater than or equal to 3 days after the last antigenic exposure required repeated mediator release, we compared dose-response changes in lung resistance (RL) to acetylcholine (ACh) in animals sensitized with 1% ovalbumin (OA), 4% Bordatella pertussis aerosol and subsequently challenged with 0.5% OA aerosol twice weekly for 4-6 wk vs. animals receiving saline aerosol instead of OA. Despite antihistamine pretreatment, each OA challenge produced cyanosis and inspiratory indrawing. Blood gas analysis in six guinea pigs revealed an immediate fall in arterial PO2 (PaO2) from 104.3 +/- 4.9 to 35.4 +/- 2.2 Torr after a 1-min exposure to aerosolized OA. ACh dose-response measurements of RL 3 days after the last OA challenge demonstrated a leftward shift and an increased magnitude of response. These differences were less marked at 7 days, and by 14 days after the last OA challenge, ACh dose-response curves were not different from those of control guinea pigs. Sensitization without repeated antigen challenge did not cause hyperresponsiveness. Morphometric analysis showed significantly increased numbers of eosinophils in the epithelium of airways in hyperresponsive guinea pigs, without neutrophil infiltration or alterations in epithelium and airway wall areas. We conclude that repeated antigenic challenge, but not sensitization alone, causes prolonged AHR in guinea pigs, which is associated with tissue eosinophilia.     

Heme oxygenase attenuates allergen-induced airway inflammation and hyperreactivity in guinea pigs

Heme oxygenase (HO), the heme-degrading enzyme, has shown anti-inflammatory effects in several models of pulmonary diseases. HO is induced in airways during asthma; however, its functional role is unclear. Therefore, we evaluated the role of HO on airway inflammation [evaluated by bronchoalveolar lavage (BAL) cellularity and BAL levels of eotaxin, PGE(2), and proteins], mucus secretion (evaluated by analysis of MUC5AC gene expression and periodic acid-Schiff staining), oxidative stress (evaluated by quantification of 4-hydroxynonenal adducts and carbonylated protein levels in lung homogenates), and airway responsiveness to histamine in ovalbumin (OVA)-sensitized and multiple aerosol OVA or saline-challenged guinea pigs (6 challenges, once daily, OVA group and control group, respectively). Airway inflammation, mucus secretion, oxidative stress, and responsiveness were significantly increased in the OVA group compared with the control group. HO upregulation by repeated administrations of hemin (50 mg/kg i.p.) significantly decreased airway responsiveness in control animals and airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These effects were reversed by the concomitant administration of the HO inhibitor tin protoporphyrin-IX (50 micromol/kg i.p.). Repeated administrations of tin protoporphyrin-IX alone significantly increased airway responsiveness in control animals but did not modify airway inflammation, mucus secretion, oxidative stress, and responsiveness in OVA animals. These results suggest that upregulation of the HO pathway has a significant protective effect against airway inflammation, mucus hypersecretion, oxidative stress, and hyperresponsiveness in a model of allergic asthma in guinea pigs.     

Blockade of airway inflammation and hyperresponsiveness by HIV-TAT-dominant negative Ras

We have reported previously that HIV-TAT-dominant negative (dn) Ras inhibits eosinophil adhesion to ICAM-1 after activation by IL-5 and eotaxin. In this study, we evaluated the role of Ras in Ag-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of dnRas, which was fused to an HIV-TAT protein transduction domain (TAT-dnRas). Uptake of TAT-dnRas (t(1/2) = 12 h) was demonstrated in leukocytes after i.p. administration. OVA-sensitization significantly increased eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid 24 h after final challenge. Treatment of animals with 3-10 mg/kg TAT-dnRas blocked the migration of eosinophils from 464 +/- 91 x 10(3)/ml to 288 +/- 79 x 10(3)/ml with 3 mg/kg of TAT-dnRas (p < 0.05), and further decreased to 116 +/- 63 x 10(3)/ml after 10 mg/kg TAT-dnRas (p < 0.01). Histological examination demonstrated that inflammatory cell infiltration (largely eosinophils and mononuclear cells) and mucin production around the airways caused by OVA were blocked by TAT-dnRas. OVA challenge also caused airway hyperresponsiveness to methacholine, which was dose dependently blocked by treatment with TAT-dnRas. TAT-dnRas also blocked Ag-induced IL-4 and IL-5, but not IFN-gamma, production in lung tissue. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by pretreatment with TAT-dnRas. By contrast, TAT-green fluorescent protein or dnRas lacking the TAT protein transduction domain did not block airway inflammation, cytokine production, or airway hyperresponsiveness. We conclude that Ras mediates Th2 cytokine production, airway inflammation, and airway hyperresponsiveness in immune-sensitized mice.     

Priming with murine recombinant interleukin-5 resulted in the augmentation of PAF-induced airway hyperresponsiveness to histamine in guinea pigs

The effects of pretreatment with murine recombinant interleukin 5 (mrIL-5) on platelet activating factor (PAF)-induced bronchoconstriction and airway hyperreactivity were investigated in guinea pigs. The intratracheal administration of mr IL-5 (2.5–10 μg) augmented platelet activating factor (PAF; 50 ng/kg)-induced bronchoconstriction in guinea pigs. When IL-5 (2.5 μg) was injected intratracheally, PAF (25 ng/kg)-induced bronchoconstriction was not affected, but PAF-induced airway hyperresponsiveness to histamine was exacerbated. Airway inflammation, in terms of increased capillary permeability and the accumulation of leukocytes in bronchoalveolar lavage fluid, was not produced by pretreatment with PAF (25 ng/kg), mrIL-5 (2.5 μg), or by a combination of these agents. This mrIL-5-induced augmentation of airway hyperreactivity by PAF was clearly inhibited by the phosphodiesterase-type III inhibitors, SDZ-MKS-492 and AH 21–132, but not by aminophylline.     

O3-induced airway hyperresponsiveness to noncholinergic system and other stimuli.

The effect of O3 exposure (3 ppm, 1 h) on the in vivo and in vitro airway responsiveness, as well as the changes in cell contents in bronchoalveolar lavage (BAL) fluid, were evaluated 16-18 h after O3 exposure in sensitized and nonsensitized male guinea pigs. The sensitization procedure was performed through repeated inhalation of ovalbumin for 3 wk. Increase in pulmonary insufflation pressure produced by the excitatory nonadrenergic noncholinergic (eNANC) system, histamine, and antigen were assessed in in vivo conditions, whereas airway responsiveness to histamine and substance P was evaluated in in vitro conditions by use of tracheal chains with or without epithelium and lung parenchymal strips. We found that O3 exposure 1) increased the neutrophil content in BAL fluids in both sensitized and nonsensitized guinea pigs, 2) caused hyperresponsiveness to eNANC stimulation in nonsensitized guinea pigs (although combination of sensitization and O3 exposure paradoxically abolished the hyperresponsiveness to eNANC stimulation), 3) increased the in vivo bronchoconstrictor responses to histamine and antigen, 4) caused hyperresponsiveness to substance P in nonsensitized tracheae with or without epithelium and in sensitized tracheae with epithelium, 5) did not modify the responsiveness to histamine in tracheae with or without epithelium (and in addition, epithelium removal caused hyperresponsiveness to histamine even in those tracheae exposed to O3), and 6) produced hyperresponsiveness to histamine in lung parenchymal strips either from sensitized or nonsensitized guinea pigs.     

Effect of platelet-activating factor on airway vascular permeability: possible mechanisms

We studied the effects of the potent inflammatory mediator, platelet-activating factor (PAF), on vascular permeability in airways (and other tissues) of guinea pigs by measuring extravasation of circulating Evans blue dye. PAF caused a dose-dependent increase in vascular permeability. At 1 ng/kg iv, PAF caused an increase in Evans blue extravasation of 220% (P less than 0.05) in the trachea, with the greatest effect at a dose of 100 ng/kg (858%; P less than 0.01). Histamine (150 micrograms/kg iv) caused a 320% increase over base line in the trachea and 200% in main bronchi; this effect was equivalent to that induced by 10 ng/kg PAF in the trachea and 1 ng/kg in main bronchi. The duration of effect of PAF was greatest in main bronchi (less than 10 min). Platelet depletion with a cytotoxic antibody, or the cyclooxygenase inhibitor, indomethacin, or the cyclooxygenase-lipoxygenase inhibitor, BW 7556, did not affect the vascular permeability response to PAF. The PAF-receptor antagonist, BN 52063, inhibited Evans blue extravasation in the airways in a dose-dependent manner, with complete inhibition at 5 mg/kg. Thus PAF-induced airway vascular leakage is mediated by specific receptors but not by products of arachidonic acid metabolism or by platelets. Increased airway microvascular leakage induced by PAF may lead to plasma extravasation and airway edema, factors that may contribute to the airway narrowing and hyperresponsiveness induced by PAF.     

Increased in vitro responses of tracheal smooth muscle from hyperresponsive guinea pigs

Repeated aerosol antigen challenge of previously sensitized guinea pigs induces airway hyperresponsiveness to inhaled acetylcholine. To determine the mechanism producing these airway changes and assuming that changes in the trachealis muscle reflect changes in muscle of the entire tracheobronchial tree, we examined the in vitro smooth muscle mechanics and morphometric parameters of tracheae from guinea pigs demonstrating hyperresponsiveness in vivo vs. tracheae from control guinea pigs. No differences between these groups were found in luminal volume at zero transmural pressure, passive pressure-volume characteristics, or area of airway wall. Smooth muscle areas were slightly less in tracheae from hyperresponsive guinea pigs. Tracheae from hyperresponsive guinea pigs had both significantly increased isovolumetric force generation and isobaric shortening compared with tracheae from controls when evaluated over the range of transmural pressures from -40 to 40 cmH2O. We conclude that the in vivo airway hyperresponsiveness induced with repeated antigen challenge is associated with both increased force generation and shortening of tracheal smooth muscle without increased muscle mass, suggesting enhanced contractile activity.     

Intratracheal priming with ovalbumin- and ovalbumin 323-339 peptide-pulsed dendritic cells induces airway hyperresponsiveness, lung eosinophilia, goblet cell hyperplasia, and inflammation

Dendritic cells (DC) are the primary APC responsible for the capture of allergens in the airways and the shuttling of processed allergens to the draining lymph nodes where Ag presentation and T cell activation take place. The mechanism of this Ag handling and presentation in asthma is poorly understood. In addition, the feasibility of asthma induction by DC priming has not been directly tested. In this report an asthma model using intratracheally (i.t.) injected splenic DC was used to address these issues. DC pulsed with a model Ag OVA or the major MHC class II-restricted OVA T epitope peptide OVA(323-339) and instilled i.t. primed mice to exhibit asthma-like diseases. With OVA as the Ag, mice exhibit airway hyperresponsiveness (AHR), lung eosinophilia and inflammation, and pulmonary goblet cell hyperplasia. In OVA(323-339)-immunized mice, AHR and goblet cell hyperplasia were noted, with little eosinophilia and parenchymal inflammation. The latter finding provides evidence for dissociating AHR from eosinophilia. In both cases mediastinal node hypertrophy occurred, and high levels of Th2 cytokines were produced by the lung and mediastinal lymph node cells (LNC). Interestingly, mediastinal LNC also produced high levels of Th1 cytokines. Lung cells produced much less Th1 cytokines than these LNC. These results demonstrate that DC when introduced i.t. are potent in inducing asthma-like diseases by recruiting lymphocytes to the lung-draining lymph nodes and by stimulating Th2 responses and also suggest that the lung environment strongly biases T cell responses to Th2.     

Length oscillation induces force potentiation in infant guinea pig airway smooth muscle

Deep inspiration counteracts bronchospasm in normal subjects but triggers further bronchoconstriction in hyperresponsive airways. Although the exact mechanisms for this contrary response by normal and hyperresponsive airways are unclear, it has been suggested that the phenomenon is related to changes in force-generating ability of airway smooth muscle after mechanical oscillation. It is known that healthy immature airways of both humans and animals exhibit hyperresponsiveness. We hypothesize that the profile of active force generation after mechanical oscillation changes with maturation and that this change contributes to the expression of airway hyperresponsiveness in juveniles. We examined the effect of an acute sinusoidal length oscillation on the force-generating ability of tracheal smooth muscle from 1 wk, 3 wk, and 2- to 3-mo-old guinea pigs. We found that the length oscillation produced 15-20% initial reduction in active force equally in all age groups. This was followed by a force recovery profile that displayed striking maturation-specific features. Unique to tracheal strips from 1-wk-old animals, active force potentiated beyond the maximal force generated before oscillation. We also found that actin polymerization was required in force recovery and that prostanoids contributed to the maturation-specific force potentiation in immature airway smooth muscle. Our results suggest a potentiated mechanosensitive contractile property of hyperresponsive airway smooth muscle. This can account for further bronchoconstriction triggered by deep inspiration in hyperresponsive airways.     

Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling

Asthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. Interleukin 13 (IL-13) is a pleiotropic cytokine produced mainly by T cells. A substantial amount of evidence suggests that IL-13 plays a critical role in the pathogenesis of asthma. Therefore, a neutralizing anti-IL-13 monoclonal antibody could provide therapeutic benefits to asthmatic patients. To test the concept we have generated a neutralizing rat anti-mouse IL-13 monoclonal antibody, and evaluated its effects in a chronic mouse model of asthma. Chronic asthma-like response was induced in ovalbumin (OVA) sensitized mice by repeated intranasal OVA challenges. After weeks of challenge, mice developed airway hyperresponsiveness (AHR) to methacholine stimulation, severe airway inflammation, hyper mucus production, and subepithelial fibrosis. When given at the time of each intranasal OVA challenge, anti-IL-13 antibody significantly suppressed AHR, eosinophil infiltration, proinflammatory cytokine/chemokine production, serum IgE, and most interestingly, airway remodeling. Taken together, these results strongly suggest that a neutralizing anti-human IL-13 monoclonal antibody could be an effective therapeutic agent for asthma.     

O3-induced mucosa-linked airway muscle hyperresponsiveness in the guinea pig.

We investigated the effects of ozone exposure (3.0 ppm, 2 h) on the responsiveness of guinea pig airway muscle in vitro from animals developing bronchial hyperreactivity. Muscarinic reactivity in vivo was determined by measuring specific airway resistance (sRaw) in response to increasing concentrations of aerosolized acetylcholine (ACh) administered before and 30 min after exposure. Immediately after reactivity testing, multiple tracheal rings from ozone- and air-exposed animals were prepared and the contractile responses to increasing concentrations of substance P, ACh, or KCl were assessed in the presence of 10 microM indomethacin with or without 1 microM phosphoramidon, an inhibitor of neutral endopeptidase. Isometric force generation in vitro was measured on stimulation by cumulative concentrations of the agonists, and force generation (in g/cm2) was calculated after determination of muscle cross-sectional area. The smooth muscle of mucosa-intact airways from guinea pigs with ozone-induced bronchial hyper-reactivity proved to be hyperresponsive in vitro to substance P and ACh but not to KCl. Pretreatment with phosphoramidon abolished the increase in substance P responsiveness but had no effect on muscarinic hyperresponsiveness after ozone exposure. Furthermore, substance P responsiveness was not augmented in ozone-exposed airways in which the mucosa had been removed before testing in vitro. Likewise, muscarinic hyperresponsiveness was not present in ozone-exposed airways without mucosa. Our data indicate that airway smooth muscle responsiveness is increased in guinea pigs with ozone-induced bronchial hyperreactivity and suggest that this hyperresponsiveness may be linked to non-cyclooxygenase mucosa-derived factors.     

Antibody-antigen interaction in the airway drives early granulocyte recruitment through BLT1

Antibody-antigen interactions in the airway initiate inflammation in acute asthma exacerbations. This inflammatory response is characterized by the recruitment of granulocytes into the airways. In murine models of asthma, granulocyte recruitment into the lung contributes to the development of airway hyperresponsiveness (AHR), mucus production, and airway remodeling. Leukotriene B4 is a mediator released following antigen challenge that has chemotactic activity for granulocytes, mediated through its receptor, BLT1. We investigated the role of BLT1 in granulocyte recruitment following antigen challenge. Wild-type mice and BLT1-/- mice were sensitized and challenged with ovalbumin (OVA) to induce acute allergic airway inflammation. In addition, to explore the relevance to antibody-antigen interactions, we injected OVA bound to anti-OVA IgG1 or anti-OVA IgE intratracheally into na?ve wild-type and BLT1-/- mice. Cell composition of the lungs, cytokine levels, histology, and AHR were determined. After sensitization and challenge with ovalbumin, there was significantly reduced neutrophil and eosinophil recruitment into the airways of BLT1-/- mice compared with wild-type animals after one or two daily antigen challenges, but this difference was not seen after three or four daily antigen challenges. Mucus production and AHR were not affected. Intratracheal injection of OVA bound to IgG1 or IgE induced neutrophil recruitment into the airways in wild-type mice but not in the BLT1-/- mice. We conclude that BLT1 mediates early recruitment of granulocytes into the airway in response to antigen-antibody interactions in a murine model of acute asthma.     

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses

Background

Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.

Methods

BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.

Results

Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.

Conclusion

These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.     

An endothelin receptor antagonist,SB-217242, inhibits airway hyperresponsiveness in allergic mice

Within the airways, endothelin-1 (ET-1) can exert a range of prominent effects, including airway smooth muscle contraction, bronchial obstruction, airway wall edema, and airway remodeling. ET-1 also possesses proinflammatory properties and contributes to the late-phase response in allergic airways. However, there is no direct evidence for the contribution of endogenous ET-1 to airway hyperresponsiveness in allergic airways. Allergic inflammation induced in mice by sensitization and challenge with the house dust mite allergen Der P1 was associated with elevated levels of ET-1 within the lung, increased numbers of eosinophils within bronchoalveolar lavage fluid and tissue sections, and development of airway hyperresponsiveness to methacholine (P < 0.05, n = 6 mice per group). Treatment of allergic mice with an endothelin receptor antagonist, SB-217242 (30 mg x kg(-1) x day(-1)), during allergen challenge markedly inhibited airway eosinophilia (bronchoalveolar lavage fluid and tissue) and development of airway hyperresponsiveness. These findings provide direct evidence for a mediator role for ET-1 in development of airway hyperresponsiveness and airway eosinophilia in Der P1-sensitized mice after antigen challenge.     

IgE-dependent mast cell activation potentiates airway responses in murine asthma models

We have studied murine models of asthma using FcepsilonRIalpha-chain-deficient (FcepsilonRIalpha(-/-)) mice to investigate the role of IgE-dependent mast cell activation in these models. When mice were either 1) immunized once with OVA in alum i.p. and then challenged with OVA intranasally, or 2) repeatedly immunized with OVA in the absence of adjuvant and subsequently challenged with nebulized OVA, FcepsilonRalpha(-/-) mice had significantly fewer eosinophils and lower IL-4 levels in their bronchoalveolar lavage fluid compared with wild-type mice. When mice were given anti-IL-5 antibody before OVA challenge in protocol 1, eosinophilic infiltration into the airways was significantly suppressed in both genotypes, but only FcepsilonRIalpha(-/-) mice showed significantly reduced airway hyperresponsiveness (AHR). In addition, when mice immunized and challenged with OVA also received a late OVA provocation at a higher concentration and were then exposed to methacholine, only wild-type mice developed a substantial increase in AHR. Since FcepsilonRI is expressed mainly on mast cells in mouse airways, we conclude that IgE-dependent activation of this cell type plays an important role in the development of allergic airway inflammation and AHR in mice. The models used may be of value for testing inhibitors of IgE or mast cells for development of therapeutic agents for human asthma.     

An essential role of mast cells in the development of airway hyperresponsiveness in a murine asthma model

Immunization of BALB/c mice with alum-adsorbed OVA, followed by three bronchoprovocations with aerosolized OVA, resulted in the development of airway hyperresponsiveness (AHR) and allergic inflammation in the lung accompanied by severe infiltration of eosinophils into airways. In this murine asthma model, administration of monoclonal anti-IL-5 Ab before each Ag challenge markedly inhibited airway eosinophilia, but the treatment did not affect the development of AHR. Immunization and aerosol challenges with OVA following the same protocol failed to induce AHR in the mast cell-deficient W/Wv mice, but induced AHR in their congenic littermates, i.e., WBB6F1 (+/+) mice. No significant difference was found between the W/Wv mice and +/+ mice with respect to the IgE and IgG1 anti-OVA Ab responses and to the airway eosinophilia after Ag provocations. It was also found that reconstitution of W/Wv mice with bone marrow-derived mast cells cultured from normal littermates restored the capacity of developing Ag-induced AHR, indicating that lack of mast cells was responsible for the failure of W/Wv mice to develop Ag-induced AHR under the experimental conditions. However, the OVA-immunized W/Wv mice developed AHR by increasing the frequency and Ag dose of bronchoprovocations. The results suggested that AHR could be developed by two distinct cellular mechanisms. One would go through mast cell activation and the other is IgE/mast cell independent but an eosinophil/IL-5-dependent mechanism.     

Decreased airway narrowing and smooth muscle contraction in hyperresponsive pigs.

Increased smooth muscle contractility or reduced smooth muscle mechanical loads could account for the excessive airway narrowing and hyperresponsiveness seen in asthma. These mechanisms were investigated by using an allergen-induced porcine model of airway hyperresponsiveness. Airway narrowing to electric field stimulation was measured in isolated bronchial segments, over a range of transmural pressures (0-20 cmH(2)O). Contractile responses to ACh were measured in bronchial segments and in isolated tracheal smooth muscle strips isolated from control and test (ovalbumin sensitized and challenged) pigs. Test airways narrowed less than controls (P < 0.0001). Test pigs showed reduced contractility to ACh, both in isolated bronchi (P < 0.01) and smooth muscle strips (P < 0.01). Thus isolated airways from pigs exhibiting airway hyperresponsiveness in vivo are hyporesponsive in vitro. The decreased narrowing in bronchi from hyperresponsive pigs may be related to decreased smooth muscle contractility. These data suggest that mechanisms external to the airway wall may be important to the hyperresponsive nature of sensitized lungs.     

The presence of LPS in OVA inhalations affects airway inflammation and AHR but not remodeling in a rodent model of asthma

Ovalbumin (OVA) is the most frequently used allergen in animal models of asthma. Lipopolysaccharide (LPS) contaminating commercial OVA may modulate the evoked airway inflammatory response to OVA. However, the effect of LPS in OVA on airway remodeling, especially airway smooth muscle (ASM) has not been evaluated. We hypothesized that LPS in commercial OVA may enhance allergen-induced airway inflammation and remodeling. Brown Norway rats were sensitized with OVA on day 0. PBS, OVA, or endotoxin-free OVA (Ef-OVA) was instilled intratracheally on days 14, 19, 24. Bronchoalveolar lavage (BAL) fluid, lung, and intrathoracic lymph node tissues were collected 48 h after the last challenge. Immunohistochemistry for α-smooth muscle actin, Periodic-Acid-Schiff staining, and real-time qPCR were performed. Airway hyperresponsiveness (AHR) was also measured. BAL fluid macrophages, eosinophils, neutrophils, and lymphocytes were increased in OVA-challenged animals, and macrophages and neutrophils were significantly lower in Ef-OVA-challenged animals. The ASM area in larger airways was significantly increased in both OVA and Ef-OVA compared with PBS-challenged animals. The mRNA expression of IFN-γ and IL-13 in lung tissues and IL-4 in lymph nodes was significantly increased by both OVA and Ef-OVA compared with PBS and were not significantly different between OVA and Ef-OVA. Monocyte chemoattractant protein (MCP)-1 in BAL fluid and AHR were significantly increased in OVA but not in Ef-OVA. LPS contamination in OVA contributes to the influx of macrophages and MCP-1 increase in the airways and to AHR after OVA challenges but does not affect OVA-induced Th1 and Th2 cytokine expression, goblet cell hyperplasia, and ASM remodeling.     

In vivo PAF-induced airway eosinophil accumulation reduces bronchial responsiveness to inhaled histamine

Chronic eosinophilic bronchitis and bronchial hyperresponsiveness have been considered to be the fundamental features of bronchial asthma. However, the role of airway eosinophils in bronchial responsiveness in vivo has not been fully discussed. The aim of this study was to investigate the direct effect of airway eosinophil accumulation on bronchial responsiveness in vivo. Guinea pigs were transnasally treated with platelet activating factor (PAF) or vehicle twice a week for a total of 3 weeks. Anesthetized guinea pigs were surgically cannulated and artificially ventilated 48 h after the last administration of PAF or vehicle. Ten minutes after the installation of artificial ventilation, ascending doses of histamine were inhaled. In a subsequent study, selective inhibitors of diamine oxidase and histamine N-methyltransferase were intravenously administered before the histamine inhalation in the PAF-treated animals. Next study was conducted 20 min after treatment with indomethacin in this study line. Finally, ascending doses of methacholine were inhaled in our animal model. Proportion of eosinophils and the number of nuclear segmentation in bronchoalveolar lavage fluid significantly increased in guinea pigs treated with PAF compared with vehicle and this finding was confirmed histologically. Nevertheless, bronchial responsiveness to inhaled histamine, but not methacholine, was significantly decreased by the PAF treatment. This bronchoprotective effect induced by PAF remained following aminoguanidine and histamine N-methyltransferase administration, but abolished by treatment of indomethacin. These results suggest that in vivo airway eosinophils may reduce nonspecific bronchial responsiveness through production of inhibitory or bronchoprotective prostanoids, but not through histaminase production.     

Recombinant human platelet-activating factor-acetylhydrolase inhibits airway inflammation and hyperreactivity in mouse asthma model

Numerous in vitro and in vivo studies in both animal models and human asthmatics have implicated platelet-activating factor (PAF) as an important inflammatory mediator in asthma. In a murine asthma model, we examined the anti-inflammatory activities of recombinant human PAF-acetylhydrolase (rPAF-AH), which converts PAF to biologically inactive lyso-PAF. In this model, mice sensitized to OVA by i.p. and intranasal (i.n.) routes are challenged with the allergen by i.n. administration. The OVA challenge elicits an eosinophil infiltration into the lungs with widespread mucus occlusion of the airways and results in bronchial hyperreactivity. The administration of rPAF-AH had a marked effect on late-phase pulmonary inflammation, which included a significant reduction in airway eosinophil infiltration, mucus hypersecretion, and airway hyperreactivity in response to methacholine challenge. These studies demonstrate that elevating plasma levels of PAF-AH through the administration of rPAF-AH is effective in blocking the late-phase pulmonary inflammation that occurs in this murine allergen-challenge asthma model. These results suggest that rPAF-AH may have therapeutic effects in patients with allergic airway inflammation.