Robert H. Gundel

Human eosinophil major basic protein induces airway constriction and airway hyperresponsiveness in primates.

We have examined the effects of direct intratracheal instillation of purified eosinophil granule proteins on pulmonary function and airway responsiveness in primates. The results of this study show for the first time that installation of major basic protein (MBP) directly into the trachea of primates results in a significant and dose-related increase in airway responsiveness to inhaled methacholine. Furthermore, MBP and eosinophil peroxidase (EPO) induce a transient bronchoconstriction immediately after instillation that resolves by 1 h postinstillation. In contrast, instillation of other eosinophil granule proteins had no effect on airway responsiveness or pulmonary function. These data indicate a direct role of the eosinophil in the pathogenesis of airway hyperresponsiveness. We suggest that the MBP of human eosinophils has an effector role in the pathogenesis of airway hyperresponsiveness which may involve active interaction with resident airway tissue cells. MBP may also mediate altered lung function in various inflammatory lung diseases associated with pulmonary eosinophilia.

Endothelial leukocyte adhesion molecule-1 mediates antigen-induced acute airway inflammation and late-phase airway obstruction in monkeys.

This study examines the role of endothelial leukocyte adhesion molecule-1 (ELAM-1) in the development of the acute airway inflammation (cell influx) and late-phase airway obstruction in a primate model of extrinsic asthma. In animals sensitive to antigen, a single inhalation exposure induced the rapid expression of ELAM-1 (6 h) exclusively on vascular endothelium that correlated with the influx of neutrophils into the lungs and the onset of late-phase airway obstruction. In contrast, basal levels of ICAM-1 was constitutively expressed on vascular endothelium and airway epithelium before antigen challenge. After the single antigen exposure, changes in ICAM-1 expression did not correlate with neutrophil influx or the change in airway caliber. This was confirmed by showing that pretreatment with a monoclonal antibody to ICAM-1 did not inhibit the acute influx of neutrophils associated with late-phase airway obstruction, whereas a monoclonal antibody to ELAM-1 blocked both the influx of neutrophils and the late-phase airway obstruction. This study demonstrates a functional role for ELAM-1 in the development of acute airway inflammation in vivo. We conclude that, in primates, the late-phase response is the result of an ELAM-1 dependent influx of neutrophils. Therefore, the regulation of ELAM-1 expression may provide a novel approach to controlling the acute inflammatory response, and thereby, affecting airway function associated with inflammatory disorders, including asthma.

Acidic polyamino acids inhibit human eosinophil granule major basic protein toxicity. Evidence of a functional role for ProMBP.

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and mammalian cells in vitro, is a single polypeptide chain rich in arginine. MBP has been localized on damaged helminths and tissues in hypersensitivity diseases including bronchial asthma. The MBP cDNA indicates that MBP is translated as a slightly acidic preproprotein with an acidic propart. To test the hypothesis that the acidic pro-part of proMBP inhibits the toxicity of mature MBP, acidic polyamino acids (aa) were used as antagonists of MBP toxicity to K562 cells and guinea pig tracheal epithelium and used as antagonists of MBP airway hyperresponsiveness in primates. The acidic poly aa inhibited MBP toxicity and MBP airway hyperresposiveness. The acidic poly aa inhibited MBP toxicity in a charge-dependent manner similar to that proposed for proMBP, suggesting that the acidic pro-part of proMBP functions to mask mature MBP toxicity. This inhibition was not limited to MBP, but also applied to polyarginine and eosinophil cationic protein. These acidic poly aa may be useful to inhibit the actions of a number of cationic toxins released by the eosinophil in numerous hypersensitivity diseases.

The role of intercellular adhesion molecule-1 in chronic airway inflammation

We have examined the role of intercellular adhesion molecule‐1 (ICAM‐1) in chronic airway inflammation and airway hyperresponsiveness in a primate model of asthma. Airway cellular composition was assessed by bronchoalveolar lavage (BAL) and airway responsiveness was measured as the bronchoconstrictor response to inhaled methacholine. In animals with chronic airway inflammation (increased BAL eosinophils) and sustained airway hyperresponsiveness, a 7 day dosing scheme with a murine anti‐human ICAM‐1 monoclonal antibody (R6.5, 2 mg/kg/day; i.v.) did not reduce the existing airway inflammation or airway hyperresponsiveness. In contrast, a similar dosing scheme with dexamethasone (0.2 mg/kg/day, i.m.) was found to significantly reduce both the airway eosinophilia and hyperresponsiveness. However, one week after cessation of dexamethasone treatment, the airway inflammation and hyperresponsiveness returned to pre‐treatment levels. In further experiments where animals were first treated with dexamethasone (7 days) followed by a 7 day treatment with R6.5, the reoccurrence of airway inflammation and subsequent increase in airway responsiveness was prevented. We conclude that the efficacy of ICAM‐1 is primarily associated with inhibition of the influx of inflammatory cells into the airways and subsequent reduction in airway responsiveness. These data suggest that in lungs with pre‐existing inflammation the modulation of ICAM‐1 following treatment with glucocorticoids may be a novel and more selective long‐term treatment for control of the chronic airway inflammation and hyperresponsiveness associated with bronchial asthma.

The role of 5-lipoxygenase products in preclinical models of asthma

BACKGROUND The action of 5-lipoxygenase on arachidonic acid generates potent inflammatory mediators that may contribute to the pathophysiology of asthma. METHODS Using the potent and selective 5-lipoxygenase inhibitor BI-L-239, we have examined the role of 5-lipoxygenase products in three animal models of asthma. RESULTS In vitro BI-L-239 inhibited 5-lipoxygenase product generation from human lung mast cells, alveolar macrophages, and peripheral blood leukocytes with a concentration that would provide 50% inhibition values of 28 to 340 nmol/L. A 36-fold selectivity for immunoreactive leukotriene C4 versus immunoreactive prostaglandin D2 inhibition was demonstrated in mast cells. In anesthetized cynomolgus monkeys, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced immunoreactive leukotriene C4 release (maximum, 73%; bronchoalveolar lavage [BAL], 20 minutes), late-phase bronchoconstriction (maximum, 41%; +6 to 8 hours), and neutrophil infiltration (maximum, 63%; BAL, +8 hours). In conscious sheep, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced late-phase bronchoconstriction (maximum, 66%; +6 to 8 hours) and increase in airway responsiveness (maximum, 82%; carbachol, +24 hours). The acute bronchoconstriction was shortened, and neutrophil infiltration diminished (maximum, 61%; BAL, +8 hours) in this model. Finally in conscious actively sensitized guinea pigs pretreated with pyrilamine and indomethacin, inhaled BI-L-239 attenuated acute bronchoconstriction (maximum, 80%; +5 to 15 minutes), leukocyte infiltration (58%; BAL, +3 days) and increase in airway responsiveness (100%; methacholine, +3 days) induced by three alternate-day ovalbumin inhalations. CONCLUSIONS In conclusion, results in these three animal models indicate that 5-lipoxygenase products may be major contributors to the bronchoconstriction (especially late phase), leukocyte infiltration, and airway hyperresponsiveness that characterize asthma.

Effects of single and multiple inhalations of antigen on airway responsiveness in monkeys

Airway hyperresponsiveness is an important and characteristic feature of asthma. In monkeys, we have reported that antigen (Ag) inhalation induces a prolonged airway eosinophilia, that chronic airway eosinophilia is associated with marked airway hyperresponsiveness, and that chronic Ag inhalations induce airway eosinophilia and hyperresponsiveness. In this study we have determined the effects of acute Ag inhalation(s) on airway responsiveness to obtain a protocol for the study of the mechanisms involved. Anesthetized and intubated adult male cynomolgus monkeys with a naturally occurring sensitivity to Ascaris suum extract were studied. Airway responsiveness (provocative concentration of nebulized and inhaled methacholine that induced a 100% increase in respiratory system resistance [Rrs] [PC100]; twofold decrease regarded as significant) and airway cell composition (bronchoalveolar lavage [BAL]) were determined 1 day before and 20 hours after a single inhalation of Ascaris extract, or 3 days before and 3 days after three alternate-day inhalations of Ascaris extract. The single inhalation of Ag (N = 7) caused an acute increase in Rrs (307% +/- 62%), an increase in BAL leukocytes, and a decrease in PC100 in three animals that was moderate (more than eightfold) in two animals. The mean +/- SE change in log PC100 was only -0.25 +/- 0.24. The multiple inhalations of Ag in the same animals caused acute increases in Rrs (178% +/- 48%, 380% +/- 83%, and 331% +/- 63%, respectively), an increase in BAL granulocytes, and a decrease in PC100 in six of seven animals (mean +/- SE change in log PC100 was -1.36 +/- 0.34) that was moderate in two and severe (more than 80-fold) in three animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of an anti-CD18 antibody (R15.7) in antigen-induced airway hyperresponsiveness (AH) and cell influx in guinea pigs

Aerosol ovalbumin challenge (OA) of sensitized guinea pigs induced airway hyperreactivity (AH) to i.v. acetylcholine (Ach) and serotonin (5-HT) 24 hr post OA. Bronchoalveolar lavage fluid 24 hrs after OA showed increased leukocytes compared to unsensitized unchallenged animals. Treatment with monoclonal antibody R15.7 (3 mg/kg i.v.,) 1 hr prior and 4 hours after OA prevented the induction of AH to Ach but not to 5-HT and reduced influx of leukocytes. We conclude: 1) antigen inhalation induces an increase in AH with an increase in proinflammatory cell influx and 2) treatment with anti-CD18 antibody inhibits cell influx and airway hyperreactivity.

The onset and recovery from airway hyperresponsiveness: relationship with inflammatory cell infiltrates and release of cytotoxic granule proteins

Previous studies from our laboratory have demonstrated a temporal relationship between eosinophil influx into the airways and the onset of airway hyperresponsiveness to inhaled methacholine. The purpose of the present study was to extend this observation by evaluating changes in airway cellular composition and measuring the levels of granulocyte‐derived mediators recovered in BAL fluid during the onset and recovery from antigen‐induced airway hyperresponsiveness. Airway cellular composition, airway responsiveness to inhaled methacholine and the levels of BAL fluid EPO and MPO were monitored over a 32 day study in eight adult male Ascaris suum sensitive cynomolgus monkeys. Repeated Ascaris suum inhalation (nine challenges during days 0–21) resulted in a selective, sustained airway eosinophilia that was temporally related with the onset and maintenance of airway hyperresponsiveness (r= 0.67, P < 0.001). The level of BAL eosinophil‐derived EPO was increased and remained elevated concurrent with the increase in airway eosinophils and airway responsiveness. During the recovery phase (days 22–32) the actual number of eosinophils remained elevated, while BAL EPO levels were significantly decreased. The recovery phase was also associated with a transient increase in the number of BAL neutrophils and MPO concentration. We conclude that the number and state of activation of airway eosinophils directly correlate with the onset and maintenance of airway hyperresponsiveness. Recovery from airway hyperresponsiveness is associated with a decrease in eosinophil activation and a transient increase in the number of activated neutrophils.

Human eosinophil major basic protein augments bronchoconstriction induced by intravenous agonists in guinea pigs

The direct effect of intratracheal (IT) administration of human major basic protein (MBP) on pulmonary inspiratory pressure (PIP), and the effect on agonist-induced change in PIP, were determined in anesthetized, ventilated guinea pigs. 500 μg MBP increased PIP from 24.1±4.3 to 49.8±7.4 cm H2O (p<0.002,n=10). Maximum PIP was achieved within 30 min after 500 μg MBP. The direct PIP response to 250 μg MBP was not different from vehicle. The PIP responses to intravenous (IV) acetylcholine (Ach) and 5-hydroxytryptamine (5-HT) were measured before and after administration of 250 μg MBP (n=12). MBP caused a modest, but significant potentiation of the increase in PIP induced by 1, 3 and 10 μg/kg Ach (24, 32 and 28%, respectively,p<0.02) and to 1 μg/kg 5-HT (43%p<0.02). We conclude that MBP at a dose that does not directly affect inspiratory pressure is capable of augmenting the PIP response to IV Ach and 5-HTin vivo.

Adhesion molecules in a primate model of allergic asthma: clinical implications for respiratory care

ConclusionPrompted by the realization of the importance of chronic airway inflammation in allergic asthma, recent interest has focused on defining the role of adhesion glycoproteins in the development of airway inflammation and subsequent changes in airway function. Many studies in vitro and in vivo have demonstrated an increased expression of certain adhesion molecules on vascular endothelium, airway epithelium and circulating leukocytes associated with the inflammatory response to allergen. Studies with animal models of allergic asthma have shown the effectiveness of antagonists of adhesion molecules in blocking the development of airway inflammation and changes in airways function. These studies suggest that inhibitors of cellular adhesion molecules may represent a novel form of treatment directed specifically at the chronic airway inflammation characteristic of bronchial asthma.