Barbara K. Stout

Activation of Human Eosinophils by Platelet-Derived Growth Factor

Activated eosinophils are believed to be major contributors to the chronic inflammatory sequelae of asthma, but the details of the mechanism of eosinophil activation in vivo are unknown. In our search for physiologically important modes of eosinophil activation, we studied the effects of recombinant human platelet-derived growth factor (PDGF) on human peripheral blood eosinophils. We compared two activation end-points: secretion of granule contents, exemplified by the release of eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and the generation of active oxygen metabolites (O2- production). PDGFc-sis dose dependently stimulated the secretion of large amounts of EPO and EDN from eosinophils. Higher concentrations of PDGF induced a dose-dependent O2- production, especially if the cells were first primed with low concentrations of phorbol ester. These activities were not seen with the AA homodimer of PDGF, suggesting that the activation was receptor dependent. However, several attempts to directly demonstrate the existence of such receptors were unsuccessful. The magnitude of the secretory response to PDGF, and the realization that eosinophils could be easily exposed to this substance as they travel towards the lung, suggests the possibility that this growth factor may be a physiologically important activator of eosinophils in the pulmonary inflammation which is associated with asthma.

Inhibition of the 5-lipoxygenase pathway with piriprost (U-60,257) protects normal primates from ozone-induced methacholine hyperresponsive small airways.

Weekly exposure to ozone in seven normal Rhesus monkeys led to induction of methacholine hypersensitive airways (RL increases 242 +/- 60% and Cdyn decreases 68 +/- 13% of baseline methacholine responses). It took 19 weeks to establish this hyperresponse that persisted for greater than 15 weeks once ozone was stopped. A second exposure led to similar response peaks in 6 weeks. At the peak of the second response, weekly 1% piriprost exposure before ozone led to a return to baseline that was not different between placebo and piriprost treated animals (9.4 +/- 1.0 and 4.3 +/- 2.9 weeks, placebo and treated, respectively P = 0.09 NS). A statistical difference in the mecholyl response in placebo and piriprost treated groups while on ozone was shown only in the Cdyn measurement (Cdyn% change 68 +/- 13 vs 24 +/- 14, placebo and piriprost, respectively P = 0.03). Off ozone (or return to baseline), a statistical difference could be detected both in RL and Cdyn (RL% changed 151 +/- 41 vs 31.1 +/- 49, P = 0.03, and for Cdyn 62.7 +/- 8 vs 9 +/- 10, P = 0.0006, placebo and piriprost, respectively). We conclude tha the primate provides a chronic model of airways reactivity in which the role of lipoxygenase is implicated because of the beneficial role of piriprost, and further that the ozone lesion is primarily in the smaller airways (possibly and alveolitis).

Activity of a Novel Hydroquinone Inhibitor of Leukotriene Synthesis (U-66,858) in the Rhesus Monkey Ascaris Reactor

The IgE-mediated hypersensitivity to Ascaris antigen in reactor rhesus primates was used to assess the pharmacologic profile of U-66,858 (1-acetoxy-2-n-butyl-4-methoxy-naphthalene). When the compound was given by the oral route, it showed dose-related inhibition of resistance (RL) and compliance (Cdyn) changes. When the compound was given by the aerosol route, it showed dose independent inhibition. In 15 animals, aerosols (52 +/- 32 to 53 +/- 10% for RL, p = 0.05 and 45 +/- 19 to 28 +/- 19% Cdyn inhibitions, p = 0.05) for 5.0-0.1% aerosol. By the oral route, inhibition was seen at 1-4 h following administration. In 5 animals, oral doses of 10 and 5 mg/kg inhibited (RL by 98 +/- 2 to 78 +/- 1.5%, p = 0.01 and Cdyn by 75 +/- 17 to 60.9 +/- 9.1%, p = 0.05) by 10 and 5 mg/kg U-66,858, respectively. The in vivo demonstration of inhibition of pulmonary bronchoconstriction by this compound, in a model known to be leukotriene sensitive, coupled with its potent in vitro inhibition of 5-lipoxygenase enzymes, suggests this compound may be of use in 5-lypoxygenase-mediated models of asthma.

Airway responses to aerosolized leukotriene D4 (LTD4) in normal and Ascaris reactor primates

Normal and Ascaris reactor primates were compared for their bronchial pulmonary response to aerosolized leukotriene D4 (LTD4). When 10 micrograms/ml LTD4 was aerosolized (total amount delivered to endotracheal tube was 1.0 micrograms) into the lungs of 6 normal primates, a small increase in total lung resistance (RL) was noted (4.4 +/- 4.5% increase, in 19 separate challenges). However, a larger effect was seen in compliance (27.6 +/- 15.8% decrease, n = 19). Ascaris reactors (n=4) demonstrated a larger RL effect than normals with almost an identical Cdyn change (RL 36.1 +/- 27.7% increase, Cdyn 32.8 +/- 18.8% decrease n = 12). When the pharmacological blockers diphenhydramine, 0.5 mg/kg and atropine, 0.5 mg/kg were administered iv separately before LTD4 challenge, significant antagonist activity was seen. Diphenhydramine inhibited the LTD4 response in normal primates (RL 64.2 +/- 44.3% and Cdyn 50.5 +/- 40.9% n = 6) and in reactors (RL 47.8 +/- 43.1% and Cdyn 19.2 +/- 20.8% n = 4). Atropine inhibited normals (RL 100% and Cdyn 73.1 +/- 32.7% n = 2) and reactors (RL 96.3 +/- 7.7 and Cdyn 47.4 +/- 35.1% n = 3). These results indicate that the LTD agonist action is partially mediated through histamine, primarily acting on lung resistance (large airways) and, in addition, may have a reflex atropine-sensitive component. The difference between the response of normal and reactor primates to LTD4 is primarily a histamine-mediated large airway response.

Late Phase Bronchoconstriction and Eosinophilia as well as Methacholine Hyperresponsiveness in Ascaris-Sensitive Rhesus Monkeys Were Reversed by Oral Administration of U-83836E

We used the Ascaris-sensitive primate model to assess the effect of oral doses of the 2-aminochroman (U-83836E) lazaroid on the antigen-induced late phase (24 h) bronchoconstriction (LPBC), eosinophilia and methacholine hyperreactivity. Following establishment of consistent lung resistance measurements and bronchoalveolar lavage eosinophilia in 4 Ascaris reactor primates, we determined the baseline aerosol mecholyl provocative challenge for 50% increase (PC50 in mg/ml) in these animals. Twenty-four hours following antigen challenge, we again determined the PC50 (mg/ml). In all 4 animals, there was a statistically significant decrease in PC50 (5.4- to 32-fold, n = 5-7; p < 0.029). Bronchoconstriction at 24 h increased in all 4 (49-86% over saline aerosol, p < 0.05). Eosinophilia increased from 21 to an average of 33% of total cells (p < 0.05 compared to saline). Repeating the antigen challenge in the presence of oral doses of 10 mg/kg U-83836E 18 and 3 h before and 6 h after challenge resulted in 53-70% inhibition of LPBC, 53-81% inhibition of eosinophilia (p < 0.05 compared to Ascaris) and return of the mecholyl PC50 (mg/ml) to before antigen levels (p = NS) thus blocking increased hyperreactivity. These results indicate U-83836E, like steroids, would be an effective drug for asthma and lung inflammation.