Invest. 1994. 93:776-787.)

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.

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.

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.

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.

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).

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The photoresection of endobronchial tumors produces smoke which is partly inhaled by the patient as well as the surgical staff. In an animal study we investigated whether a single exposure or repetitive exposures to smoke might have harmful side effects on the airways. Eleven sheep were exposed to smoke produced by laser-vaporizing (6,500 J) sections of bronchial tissue (1 cm3) in a Plexiglas chamber. The smoke analysis revealed 0.92 mg/liter particles with a mean particle size of 0.54 micron. Carbon monoxide content was estimated as 0.04%. We measured the effects of one or three separate ten-minute exposures on airway resistance, gas exchange, and mucociliary clearance rate in the trachea. We found that the smoke inhalation resulted in a decrease of arterial PO2 with relatively little change in airway mechanics. Tracheal mucus velocity, a marker of lung mucociliary clearance, was significantly depressed in a dose-dependent manner with increasing smoke exposures. Results of bronchoalveolar lavages performed before and one day after the exposure showed that the smoke inhalation induced a severe inflammation with dramatic increases of inflammatory cells. The total number of cells per milliliter lavage return increased from 3.2 million to 25 million; percent neutrophils increased from 2.3 to 45.6% and percent macrophages decreased from 86 to 41%. These findings indicate that the side effects of smoke inhalation during intrabronchial laser surgery should not be neglected. The impairment of the defense mechanism of the lung combined with the inflammation as well as hypoxia might be fatal in compromised patients. Effective smoke removal devices should be developed to protect the patient as well as the surgeon.