A small-molecule, tight-binding inhibitor of the integrin alpha(4)beta(1) blocks antigen-induced airway responses and inflammation in experimental asthma in sheep.

The leukocyte integrin very late antigen-4 (alpha(4)beta(1), CD49d/CD29) is an adhesion receptor that plays an important role in allergic inflammation and contributes to antigen-induced late responses (LAR) and airway hyperresponsiveness (AHR). In this study, we show that single doses of a new small-molecule, tight-binding inhibitor of alpha(4), BIO-1211, whether given by aerosol or intravenously, either before or 1.5 h after antigen challenge blocks allergen- induced LAR and post-antigen-induced AHR in allergic sheep. Multiple treatments with doses of BIO-1211 that were ineffective when given singly, were protective. BIO-1211 also provided dose-dependent inhibition of the early airway response (EAR) to antigen. In conjunction with the functional protection against the antigen-induced LAR and AHR, sheep treated with BIO-1211 before challenge showed significantly reduced: (1) numbers of eosinophils in bronchoalveolar lavage (BAL), (2) BAL levels of the inflammatory marker tissue kallikrein, and (3) numbers of inflammatory cells (lymphocytes, eosinophils, metachromatic staining cells, and neutrophils) in bronchial biopsies obtained after challenge when compared with corresponding biopsies after vehicle treatment. More importantly, we show for the first time that an inhibitor of alpha(4) was able to reverse post-antigen-induced AHR, thereby decreasing the time of recovery from the normal period of > 9 d to 3 d. Our results show that effective inhibition of antigen-induced airway responses can be achieved with single doses of a potent small-molecule inhibitor of alpha(4) and that such agents may be used therapeutically, as well as prophylactically, to alleviate allergen- induced inflammatory events. These data provide further support and extend the evidence for the role of alpha(4) integrins in the pathophysiologic events that follow airway antigen challenge.

Altered contractile sensitivity of isolated bronchial artery to phenylephrine in ovalbumin-sensitized rabbits.

We tested the hypothesis that atopy and/or allergic lung inflammation enhances alpha1-adrenoceptor-mediated contractions of the bronchial artery. Bronchial arterial resistance vessels were isolated from rabbits that had undergone either systemic ovalbumin (OVA) sensitization followed by saline aerosol challenge (OVA/saline rabbits), or OVA sensitization followed by OVA aerosol challenge (OVA/OVA rabbits), or no sensitization followed by saline aerosol challenge (control rabbits). In OVA/OVA rabbits, bronchoalveolar lavage and lung histology revealed lymphocytic and eosinophilic inflammation. Arterial rings were contracted with phenylephrine (PE). In endothelium-intact arteries isolated from OVA/saline and OVA/OVA rabbits, PE responsiveness was enhanced compared with that of arteries isolated from controls. The nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester increased the contractile response to PE in all three experimental groups to a similar degree, suggesting that depressed NOS activity was not involved in the enhanced PE responsiveness in OVA/saline and OVA/OVA rabbits. After endothelium removal, arteries from OVA/saline and control rabbits showed similar PE responsiveness, indicating that the enhancement of PE responsiveness was endothelium dependent, possibly due to an endothelial constricting factor. In OVA/OVA rabbits, endothelium-denuded arteries showed decreased PE responsiveness compared with the other two groups; this difference was abolished by NG-nitro-L-arginine methyl ester. We conclude that systemic sensitization with OVA per se enhances PE-induced contractions of isolated bronchial arteries in rabbits by an endothelium-dependent mechanism and that allergic lung inflammation attenuates this effect by increased nonendothelial NOS activity.

Blockade of late-phase airway responses and airway hyperresponsiveness in allergic sheep with a small-molecule peptide inhibitor of VLA-4.

The leukocyte integrin very late antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) is an adhesion receptor predominantly expressed on lymphocytes, monocytes, and eosinophils, but not on neutrophils. Recent studies with monoclonal antibodies against VLA-4 suggest that antigen-induced late responses and airway hyperresponsiveness (AHR) may depend on the recruitment and/or activation of VLA-4-expressing leukocytes. To further test this hypothesis, we administered by aerosol either a potent small-molecule inhibitor of VLA-4, which prevents VLA-4-mediated binding to fibronectin (CS-1 ligand mimic), or an inactive control (30 mg twice daily for 3 d, and on the fourth day 0.5 h before and 4 h after antigen challenge) to six sheep with airway hypersensitivity to Ascaris suum antigen. Treatment with the small-molecule VLA-4 inhibitor resulted in a significant decrease in the early antigen-induced bronchial response (40%, p < 0.05), and almost complete blockade of the late-phase airway response (88%, p < 0.05). Moreover, at 24 h after antigen challenge, AHR to inhaled carbachol was not observed when the animals were dosed with the small-molecule VLA-4 inhibitor. In accord with protection against the functional abnormalities associated with antigen challenge, analysis of biopsy specimens taken 24 h after challenge indicated that the total numbers of VLA-4-positive cells (lymphocytes, eosinophils, and metachromatic-staining cells) in the group treated with the VLA-4 inhibitor did not increase, whereas these cells increased in the control group. The active agent, but not the inactive control, significantly blocked macrophage adherence to fibronectin (FN), indicating that the CS-1 ligand interfered with VLA-4-mediated adhesion in sheep cells. These results support our previous findings with a monoclonal antibody to VLA-4, and demonstrate that a small-molecule VLA-4 inhibitor, when given by aerosol, has a protective effect against antigen-induced late responses and AHR in allergic sheep.

Norepinephrine-induced contraction of isolated rabbit bronchial artery: role of alpha 1- and alpha 2-adrenoceptor activation.

The contractile effect of norepinephrine (NE) on isolated rabbit bronchial artery rings (150-300 microns in diameter) and the role of alpha 1- and alpha 2-adrenoceptors (AR) on smooth muscle and endothelium were studied. In intact arteries, NE increased tension in a dose-dependent manner, and the sensitivity for NE was further increased in the absence of endothelium. In intact but not in endothelium-denuded arteries, the response to NE was increased in the presence of both indomethacin (Indo; cyclooxygenase inhibitor) and NG-nitro-L-arginine methyl ester [L-NAME; nitric oxide (NO) synthase inhibitor], indicating that two endothelium-derived factors, NO and a prostanoid, modulate the NE-induced contraction. The alpha 1-AR antagonist prazosin shifted the NE dose-response curve to the right, and phenylephrine (alpha 1-AR agonist) induced a dose-dependent contraction that was potentiated by L-NAME or removal of the endothelium. The sensitivity to NE was increased slightly by the alpha 2-AR antagonists yohimbine and idazoxan, and this effect was abolished by Indo or removal of the endothelium. Similarly, contractions induced by UK-14304 (alpha 2-AR agonist) were potentiated by Indo or removal of the endothelium. These results suggest that NE-induced contraction is mediated through activation of alpha 1- and alpha 2-ARs on both smooth muscle and endothelium. Activation of the alpha 1- and alpha 2-ARs on the smooth muscle causes contraction, whereas activation of the endothelial alpha 1- and alpha 2-ARs induces relaxation through release of NO (alpha 1-ARs) and a prostanoid (alpha 2-ARs).

Alpha 4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.

Eosinophils and T lymphocytes are thought to be involved in allergic airway inflammation. Both cells express the alpha 4 beta 1-integrin, very late antigen-4 (VLA-4, CD49d/CD29); alpha 4-integrins can promote cellular adhesion and activation. Therefore, we examined the in vivo effects of a blocking anti-alpha 4 monoclonal antibody, HP 1/2, on antigen-induced early and late bronchial responses, airway hyperresponsiveness, inflammatory cell influx, and peripheral leukocyte counts in allergic sheep. Sheep blood lymphocytes, monocytes, and eosinophils expressed alpha 4 and bound HP 1/2. In control sheep, Ascaris antigen challenge produced early and late increases in specific lung resistance of 380 +/- 42% and 175 +/- 16% over baseline immediately and 7 h after challenge, respectively, as well as airway hyperresponsiveness continuing for 14 d after antigen challenge. Treatment with HP 1/2 (1 mg/kg, i.v.) 30 min before antigen challenge did not affect the early increase in specific lung resistance but inhibited the late-phase increase at 5-8 h by 75% (P < 0.05) and inhibited the post-antigen-induced airway hyperresponsiveness at 1, 2, 7, and 14 d (P < 0.05, for each time). Intravenous HP 1/2 given 2 h after antigen challenge likewise blocked late-phase airway changes and postchallenge airway hyperresponsiveness. Airway administration of HP 1/2 (16-mg dose) was also effective in blocking these antigen-induced changes. Response to HP 1/2 was specific since an isotypic monoclonal antibody, 1E6, was ineffective by intravenous and aerosol administration. Inhibition of leukocyte recruitment did not totally account for the activity of anti-alpha 4 antibody since HP 1/2 neither diminished the eosinopenia or lymphopenia that followed antigen challenge nor consistently altered the composition of leukocytes recovered by bronchoalveolar lavage. Because airway administration of HP 1/2 was also active, HP 1/2 may have inhibited cell activation. Reduction of platelet-activating factor-induced eosinophil peroxidase release from HP 1/2-treated eosinophils supports such a mechanism. These findings indicate a role for alpha 4-integrins in processes that lead to airway late phase responses and persisting airway hyperresponsiveness after antigen challenge.

Oxygen radicals contribute to antigen-induced airway hyperresponsiveness in conscious sheep.

We previously showed that oxygen radicals can induce airway hyperresponsiveness (AHR) in allergic sheep. The purpose of this study was to determine whether antigen challenge results in the generation of free oxygen radicals and if these radicals contribute to antigen-induced AHR. We first determined baseline airway responsiveness in seven Ascaris suum-sensitive sheep by calculating the cumulative provocative concentration of carbachol in breath units (BU; one BU defined as one breath of a 1% wt/vol carbachol solution) that increased specific lung resistance (SRL) 400% over baseline (PC400). On a different day, the sheep underwent inhalation challenge with A. suum antigen, SRL was measured before and immediately after challenge and then hourly for 2 h, at which time SRL had returned to baseline. The postchallenge PC400 was then measured. This procedure was repeated on separate occasions, each at least 14 days apart, except that the sheep were treated with an aerosol of catalase (CAT; 38 mg in 3 ml deionized water), the enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2), at three different times: Trial 1, before antigen and then every 30 min after antigen challenge for 2 h; Trial II, 1 and 2 h after antigen challenge; and Trial III, only at 2 h after antigen challenge. In the control trial, antigen challenge caused a transient (mean +/- SEM) 303 +/- 48% increase in SRL over baseline (p < 0.05), and 2 h later, PC400 was reduced to 11.0 +/- 1.7 BU from a prechallenge value of 24.8 +/- 1.9 BU (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The 5-lipoxygenase inhibitor zileuton blocks antigen-induced late airway responses, inflammation and airway hyperresponsiveness in allergic sheep.

Leukotrienes are thought to be involved in allergen-induced airway responses. To test this hypothesis we used a newly described 5-lipoxygenase inhibitor, zileuton, and examined its effect on antigen-induced early and late bronchial responses, airway inflammation and airway hyperresponsiveness in allergic sheep. Early and late responses were determined by measuring specific lung resistance (SRL) before and serially for 8 h after antigen challenge. Airway inflammation was assessed by bronchoalveolar lavage performed before, 8 h after and 24 h after antigen challenge. Airway responsiveness was measured before and 24 h after challenge by determining the dose of inhaled carbachol that caused a 400% increase in SRL (PD400%). The sheep (n = 8) were challenged with Ascaris suum antigen once after vehicle treatment (methylcellulose) and once after treatment with zileuton (10 mg/kg in methylcellulose, p.o.) given 2 h before antigen challenge. Trials were separated by at least 21 days. Zileuton had no effect on the early bronchoconstrictor response to antigen but the drug inhibited the late bronchial response by 55% (P less than 0.05). Unlike the control trial, there was no significant increase in bronchoalveolar lavage eosinophils at 8 h post challenge in the zileuton-treated sheep. Furthermore, zileuton treatment blocked (P less than 0.05) the airway hyperresponsiveness seen 24 h after challenge. Ex vivo formation of leukotriene B4 was inhibited over several hours after a single oral dose of zileuton, indicating that the compound was acting as a 5-lipoxygenase inhibitor in vivo. These results suggest that 5-lipoxygenase metabolites contribute to allergen-induced late responses, airway inflammation and airway hyperresponsiveness in this animal model of asthma.

A bradykinin antagonist modifies allergen-induced mediator release and late bronchial responses in sheep.

We assessed the role of bradykinin (BK) in allergen-induced early and late bronchial responses, airway inflammation, mediator release, and antigen-induced airway hyperresponsiveness in allergic sheep by studying the effects of the BK B2 receptor antagonist, NPC-567 (D-Arg-[Hyp3, D-Phe7]-BK), on these parameters. Antigen challenge was performed on two occasions greater than 3 wk apart, once with placebo (control) and once after high-dose (10 mg/ml) and low-dose (5 mg/ml) treatments with aerosol NPC-567. In the control trials (n = 14) antigen challenge resulted in an early and late increase in specific lung resistance (SRL). The early response was associated with increases (p less than 0.05) in prostaglandin (PG) D2, immunoreactive kinin, tosyl-L-arginine methyl ester (TAME)-esterase, and PGE2 in bronchoalveolar lavage (BAL) fluid. The late response was associated with increases (p less than 0.05) in leukotrienes (LT) B4 and C4, thromboxane (TX) B2, 6-keto-PGF10, and PGE2. There was a significant influx of neutrophils in the BAL fluid during the late response, and airway hyperresponsiveness to carbachol aerosol was apparent 4 h after challenge. In six sheep the high-dose NPC-567 treatment (given before, during, and 4 h after antigen challenge) did not attenuate the early bronchoconstrictor response or the early release of mediators but caused a significant reduction in the late response (p less than 0.05). This protective effect was accompanied by reductions (p less than 0.05) in both the concentrations of all the mediators associated with the late response and the severity of the BAL neutrophilia. High-dose NPC-567 did not attenuate the airway hyperresponsiveness or the cellular inflammatory response seen 24 h after challenge. In eight sheep treated with the low dose of NPC-567 (given before, during, and 4, 8, and 24 h after challenge) the early response was not blocked but the late response was again inhibited, as were the mediators associated with the late response. At the low dose the drug did not prevent the airway inflammation at 8 or 24 h. The additional treatments did, however, prevent the 24 h hyperresponsiveness. These data suggest that kinin generation during antigen-induced airway anaphylaxis may be important for controlling the release of arachidonic acid metabolites from airway inflammatory cells that contribute to the development of the late response in the allergic sheep model.

Effects of P. aeruginosa-derived bacterial products on tracheal ciliary function: role of O2 radicals.

The purpose of this investigation was to evaluate the effects of bacterial products derived from Pseudomonas aeruginosa on the function of airway cilia and to assess the role of phagocytes and oxygen radicals in the observed responses. Ciliary beat frequency (CBF) was measured in a perfusion chamber with a microscopic technique using tracheal epithelial cells obtained from normal sheep by brush biopsy (70% epithelial cells, 18% macrophages, 11% neutrophils). Baseline CBF ranged between 678 and 1,126 min-1. After 20 min of perfusion with the cell free supernatant of P. aeruginosa culture (mucoid strain), a concentration-dependent depression of CBF was observed with a 58% inhibition at a 1:1 dilution (P less than 0.05). The P. aeruginosa-derived products pyocyanin and 1-hydroxyphenazine also decreased CBF in a dose-related fashion. The cilion-inhibitory effects of the supernatant and bacterial products were markedly attenuated after centrifugation of the brush preparation (80% epithelial cells, 16.5% macrophages, 3.5% neutrophils). Glucose/glucose oxidase also caused a rapid, concentration-dependent cilioinhibition or ciliostasis. Catalase blocked or attenuated the ciliary effects of the supernatant, bacterial products and glucose/glucose oxidase. Thus bacterial products released from P. aeruginosa impaired ciliary activity by a pathway which involved neutrophils and was mediated by toxic oxygen radicals.

Cellular LTB4 production differs in allergic sheep with and without late airway responses.

We tested the hypothesis that allergic sheep that develop both early and late airway responses to inhaled Ascaris suum antigen (late responders) have an increased capacity to generate leukotrienes (LTs) compared with allergic sheep that show only early responses to inhaled antigen (acute responders). To test this hypothesis, we measured LTB4 production, in vitro, by granulocytes isolated from peripheral blood and by macrophages isolated from bronchoalveolar lavage (BAL) from both groups of sheep greater than or equal to 2 wk after the animal's last antigen challenge; LTB4 production by granulocytes isolated from BAL from both groups of sheep 6 and 48 h after local airway challenge with A. suum antigen was also measured. LTB4 production was induced by incubating cells (i.e., either granulocytes or macrophages) with calcium ionophore (A23187, 2 microM) and arachidonic acid (30 microM). LTB4 production was quantitated by high-performance liquid chromatography and verified by radioimmunoassay (RIA). On stimulation peripheral blood granulocytes from late responders (n = 7) produced (means +/- SD/10(6) cells) 13.3 +/- 5.2 ng LTB4 compared with 5.3 +/- 1.5 ng LTB4 (P less than 0.05) for acute responders (n = 7). This increased LTB4 production did not result from variations in granulocyte differential or cyclooxygenase activity (as indicated by RIA measurements of prostaglandin E2 production).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ozone on the postnatal development of lamb mucociliary apparatus.

We determined whether exposure to O3 early in the postnatal period impairs the normal development of the mucociliary apparatus in lambs and whether such changes lead to prolonged abnormalities in mucociliary function. Lambs were exposed to air (controls) or to 1 ppm O3 for 4 h/day for 5 days during the 1st wk of life. Tracheal mucus velocity (TMV), a marker of lung mucociliary clearance, was measured in vivo at birth (0 wk) and up to 24 wk later, and tracheal secretory function was measured (in vitro) and the morphology of the tracheal mucosa was determined at 0 and 2 wk in both groups. In the control group, TMV increased 94% from 0 to 2 wk (P less than 0.05), continued to increase until reaching a plateau at 8 wk, and then remained constant from 8 to 24 wk. In contrast, O3-exposed lambs showed a 24% decrease in TMV from 0 to 2 wk (P less than 0.05 vs. control), and throughout the remaining time TMV remained below (P less than 0.05) that observed in control lambs. O3 exposure partially prevented the age-dependent decrease in basal secretion of tracheal macromolecules normally observed between 0 and 2 wk. These changes in secretory function were associated with a significant increase in tissue conductance (37%, P less than 0.05 vs. 0 wk), predominantly the result of active chloride secretion. The functional changes induced by O3 were associated with a retardation of the normal morphological development of the tracheal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ozone on lamb tracheal mucosa. Quantitative glycoconjugate histochemistry.

Whether or not the previously reported O3-induced abnormality in the postnatal development of tracheal secretory function in lambs is accompanied by changes in epithelial cell populations and their glycoconjugate composition was determined. Six lambs were killed at birth and 12 lambs at age 2 weeks. Of the latter 12, six were exposed to O3 (1 ppm, 4 hours daily for 5 days during the 1st week of life) and five had air-sham exposures (controls). Tracheal glycoconjugates were localized in situ with lectins to detect N-acetyl-galactosamine (galNAc), alpha-D-galactose (alpha-gal), beta-D-gal(1----3)-galNAc (beta-gal), and fucose (fuc). Mean (+/- SD) epithelial cell density (cells/mm basal lamina) was 418 +/- 57 in the newborns, 385 +/- 63 in controls (P was not significant), and 342 +/- 47 in O3-exposed lambs (P less than 0.05). Mucous cell density was 87 +/- 12 in newborns, 63 +/- 10 in controls (P less than 0.05), and 76 +/- 10 in O3 exposed lambs (P was not significant). Ciliated cells remained unchanged from birth to 2 weeks (P was not significant), but decreased (P less than 0.05) in O3-exposed lambs. All counted mucous cells contained fuc and galNAc at birth and retained these residues after sham and O3 exposure. The alpha-gal-containing mucous cells declined from 97 +/- 13 to 7 +/- 1 (P less than 0.05) and beta-gal containing cells from 39 +/- 5 to 25 +/- 4 in controls. In contrast, cells containing alpha-gal 71 +/- 10 remained at newborn levels (97 +/- 13) and beta-gal-containing cells increased from 40 +/- 5 at birth to 58 +/- 8 in O3-exposed animals (P less than 0.05). It was concluded that early postnatal exposure of lambs to O3 causes a decrease in epithelial cell density, but retards the developmental decrease in the number of tracheal mucous cells and alters the lectin detectable carbohydrate composition of mucus in these cells. These developmental defects were interpreted to be the morphologic correlates of the previously shown effects of O3 on the maturation of secretory function and mucus transport.

Developmental changes in the tracheal mucociliary system in neonatal sheep.

We studied the postnatal development of the tracheal epithelium and mucociliary system in neonatal sheep. Secretion of macromolecules (radiolabeled with 35SO4 and [3H]-threonine), unidirectional fluxes of Cl-, Na+, and water (measured with radioactive tracers), and ciliary beat frequency (CBF) were measured in tracheal tissues in vitro. Tracheal mucus transport velocity (TMV) was measured in vivo. Sheep were studied at 0, 2, 4, 8, and greater than 24 (adult) wk after birth. In newborn sheep trachea, secretion of macromolecules was significantly elevated (cf. adults), and there was basal net secretion of Cl- under short-circuit and open-circuit conditions. This induced open-circuit secretion of Na+. Secretion of macromolecules decreased rapidly by 2 wk (by 40-50%) and was not different from adult values by 4 wk. Active Na+ absorption developed rapidly, and from 2 wk onward it predominated under open-circuit conditions, inducing net Cl- absorption. These changes in secretory function were associated with an age-related increase in TMV, whereas inherent tracheal CBF was unchanged. In sheep, therefore, the newborn's trachea has elevated secretion of macromolecules and secretes Cl- and liquid under basal conditions. Normal secretory function (a reduction in secretion of macromolecules coupled with net absorption of ions and presumably of liquid also) approaches adult function by 2-4 wk of age.

A PAF antagonist blocks antigen-induced airway hyperresponsiveness and inflammation in sheep.

We studied the effects of WEB-2086, a specific antagonist of platelet-activating factor (PAF), on the development of antigen-induced airway hyperresponsiveness and inflammation in sheep (n = 8). For these studies, airway responsiveness was determined from slopes of carbachol dose-response curves (DRC) performed at base line (prechallenge) and 2 h after Ascaris suum antigen challenges in the following three protocols: 1) antigen challenge alone (control trial), 2) WEB-2086 (1 mg/kg iv) given 30 min before antigen challenge (WEB pretreatment), and 3) WEB-2086 given 2 h after antigen challenge, immediately before the postchallenge DRC (WEB posttreatment). Airway inflammation was assessed by bronchoalveolar lavage (BAL) before antigen challenge and after the postchallenge DRC for each trial. A. suum challenge resulted in acute increases in specific lung resistance that were not different among the three trials. Antigen challenge (control trial) caused a 93% increase (P less than 0.05) in the slope of the carbachol DRC when compared with the prechallenge value. WEB pretreatment (1 mg/kg) reduced (P less than 0.05) this antigen-induced hyperresponsiveness, whereas pretreatment with a 3-mg/kg dose completely prevented it. WEB posttreatment was ineffective in blocking this hyperresponsiveness. BAL neutrophils increased after antigen challenge in the control trial and when WEB-2086 was given after antigen challenge (P less than 0.05). Pretreatment with WEB-2086 (1 or 3 mg/kg) prevented this neutrophilia. This study provides indirect evidence for antigen-induced PAF release in vivo and for a role of endogenous PAF in the modulation of airway responsiveness and airway inflammation after antigen-induced bronchoconstriction in sheep.

Cellular markers of inflammation in the airways of allergic sheep with and without allergen-induced late responses.

Allergic sheep respond to inhaled Ascaris suum antigen either with an acute bronchoconstriction alone (acute responders, AR) or both an acute and late bronchoconstriction (dual responders, DR). In this study, we determined if: (1) inflammatory cell composition of bronchoalveolar lavage (BAL) obtained during the late response differs between DR and AR; (2) the difference in inflammatory cells is dependent on the prechallenge BAL cell composition; and (3) drugs that block late airway responses also modify this airway inflammation. Antigen challenge caused significant immediate mean increases in specific lung resistance (SRL) both in DR (n = 28) and in AR (n = 14), but only DR had a late increase in SRL. There were no differences between the two groups in total cell returns or in the percentage of neutrophils in BAL 7.5 to 8 h after challenge, but DR had a 3.5-fold increase (p less than 0.05) in the percentage of eosinophils. Methylprednisolone succinate (15 mg/kg intravenously) given to DR (n = 7) 3 h after antigen challenge blocked the late airway response and the eosinophil response. When BAL was performed both before and after (i.e., 7.5 to 8 h) antigen challenge, similar results were observed: AR (n = 7) and DR (n = 14) exhibited characteristic airway responses. No significant differences in prechallenge BAL cell composition were observed between AR and DR; after challenge both groups showed increases in neutrophils, but only the DR showed an increase (p less than 0.05) in eosinophils. Pretreatment of DR with the antiallergic agents (cromolyn sodium or nedocromil sodium aerosol, 20 mg) blocked the immediate and late responses and the late increase in BAL eosinophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial pneumonia stimulates macromolecule secretion and ion and water fluxes in sheep trachea.

In vivo instillation of Pasteurella haemolytica (greater than or equal to 10(7) colony-forming units/kg) into a lobar bronchus of sheep produced bacterial pneumonia by 7 days postinoculation. Infection was verified bacteriologically and histologically. Macromolecule secretion and ion and water fluxes were subsequently measured in tracheal tissues in vitro and were compared with values from sham-infected sheep. Macromolecules were radiolabeled with 35SO4 and [3H]threonine, and we measured the secretion of macromolecule-bound radiolabel onto the mucosa. Unidirectional fluxes of Cl-, Na+, and water were measured with radioactive tracers under open-circuit and short-circuit conditions. Lung infection increased basal secretion of bound 35SO4 (by 189%) and bound [3H]-threonine (by 110%). It significantly increased net Na+ absorption under open- and short-circuit conditions and induced open-circuit net absorption of Cl- and water (16 +/- 29 microliters X cm-2 X h-1). These changes were associated with specific recruitment of neutrophils and elevated levels of arachidonate metabolites (thromboxane B2 and leukotriene B4) in the airways. Thus the bacterial pneumonia-induced changes in tracheal mucus secretion may be the result of airway inflammation.

Indomethacin and FPL-57231 inhibit antigen-induced airway hyperresponsiveness in sheep.

We compared the development of antigen-induced airway hyperresponsiveness (AHR) 24 h after challenge with Ascaris suum antigen in allergic sheep with acute (n = 7) and with dual (n = 7) airway responses and then attempted to modify this AHR. Cholinergic airway responsiveness was determined by measuring the carbachol dose required to increase specific lung resistance (sRL) 150% (i.e., PC150). Subsequently the sheep were challenged with antigen and sRL was measured at predetermined times to document the presence or absence of a late response. PC150 was redetermined 24 h later followed by bronchoalveolar lavage (BAL) to assess inflammation. Only dual responders developed AHR (PC150 decreased, P less than 0.05). There were no significant differences in BAL between the two groups. Six dual responders were then, on separate occasions (greater than or equal to 3 wk), pretreated with placebo, indomethacin (2 mg/kg iv), or a leukotriene antagonist, FPL-57231 (30 mg inhaled). Neither agent significantly affected the acute response to antigen. Only FPL pretreatment blocked the late response, but both agents blocked the antigen-induced AHR 24 h later. BAL at 24 h showed no significant differences. These results indicate that only dual responders develop AHR 24 h after antigen challenge. This AHR appears independent of the late increase in sRL or the severity of pulmonary inflammation. AHR appears to be sensitive to agents that interfere with the early release or actions of cyclooxygenase and lipoxygenase metabolites in dual responders.

Effects of 0.5 ppm ozone on glycoprotein secretion, ion and water fluxes in sheep trachea.

We studied the effects of ozone (O3) exposure on airway mucus secretion. Sheep were exposed in vivo to 0.5 ppm O3, 4 h/day for 2 days (acute, n = 6), 6 wks (chronic, n = 6) or 6 wks + 1 wk recovery (chronic + recovery, n = 6). Secretion of glycoproteins (radiolabeled with 35SO4 and [3H]threonine), and transepithelial fluxes of Cl-, Na+ and water were subsequently measured in tracheal tissues in vitro, and were compared with values from control, unexposed sheep (n = 8). Acute O3 exposure increased basal secretion of sulfated glycoproteins (P less than 0.05), but had no effect on ion fluxes. Chronic exposure reduced basal glycoprotein secretion, but increased net Cl- secretion. Under open-circuit conditions, chronic exposure also induced net water secretion (P less than 0.05). With 7 days recovery, basal glycoprotein secretion (predominantly sulfated) was greatly increased above control, while the increased net secretion of Cl- and of water persisted (P less than 0.05). Histology of the airways indicated that acute exposure induced moderate hypertrophy of submucosal glands in the lower trachea (P less than 0.05), while chronic exposure (with and without recovery) induced a large hypertrophy of submucosal glands in both upper and lower trachea (P less than 0.05). Without recovery, however, the gland cells were devoid of secretory material, whereas with recovery they were full of secretory material. This suggests that the decreased glycoprotein secretion with chronic exposure alone resulted from incomplete replenishment of intracellular stores after 6 wks of stimulation. We conclude that both short- and long-term O3 exposure causes airway-mucus hypersecretion.

Impairment of tracheal mucociliary clearance but not ciliary beat frequency by a combination of low level ozone and sulfur dioxide in sheep.

We used conscious sheep to determine the effect of a combined exposure to ozone (O3) and sulfur dioxide (SO2) on tracheal mucus velocity (TMV) and ciliary beat frequency (CBF). TMV was measured in vivo by tracking the movement of radiopaque particles which had been deposited on the tracheal mucosa. CBF of tracheal epithelial cells, obtained by brushing, was measured in vitro with phase contrast microscopy. On two separate occasions, six sheep were exposed by mask to either a combination of 0.3 ppm O3 and 3 ppm SO2 or air (control) for 5 h on each of three consecutive days. TMV and CBF were measured prior to the first exposure, and immediately and 24 h after the last exposure. The combination of O3 and SO2 depressed mean TMV by 40% immediately after and by 25%, 24 h after exposure. Both values were different from the corresponding values after air exposure (p less than 0.05). However, this depression in TMV was not associated with an impairment in CBF. We conclude that in conscious sheep, exposure to a combination of low level O3 and SO2 causes a depression in TMV, and changes in CBF are probably not a primary cause of this mucociliary dysfunction.

Airway cell changes in tracheal lavage of sheep after ozone exposure.

We were interested in whether ozone (O3) could stimulate the migration of mast cells into the tracheal lumen. To test this we determined the effect of an acute O3 exposure on the types and relative numbers of cells recovered by tracheal lavage. Seven conscious sheep were intubated with an elongated nasotracheal tube. The trachea between the larynx and the cuff of the tracheal tube (15-20 cm) was lavaged repeatedly with 10-15-ml aliquots (total 350 ml) of 0.9% buffered (pH 7.4 saline, which contained the mast cell-stabilizer disodium cromoglycate (10 micrograms/ml). One hour after a baseline lavage, the sheep were exposed on separate occasions to either air (control) or 0.5 ppm O3 for 2 h. Lavages were repeated 24 h later. Cells were recovered from the lavage effluent by centrifugation across a saline/Ficoll Paque gradient. From part of this material we estimated total cells and total viable cells (with Trypan blue). The rest of the material was recentrifuged at 400 X g for 5 min, and cytological slides were made from the cell pellet. Slides were stained with Polichrome and Wright-Giemsa, and were analyzed by light microscopy. The percentages of epithelial cells, macrophages, lymphocytes, and mast cells were determined from a total count of 500 cells/slide. Differences in cell percentages between pre- and postexposure were calculated both for air and O3 exposures, and these differences were compared. Exposure to O3 resulted in an increased number of mast cells and lymphocytes when compared to the changes observed with air. It seems likely that the increase in number of luminal mast cells and lymphocytes following O3 exposure signals an enhanced inflammatory response and that these changes could contribute to O3-induced increased nonspecific airway hyperresponsiveness and susceptibility to allergic IgE-mediated airway reactions.