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Inhibition of PAF-induced eosinophil accumulation in pulmonary airways of guinea pigs by anti-asthma drugs.

Intraperitoneal (i.p.) injection of platelet activating factor (PAF) in guinea pigs caused a dose-related increase in the number of eosinophils recovered from bronchoalveolar lavage fluid (BALF). The prevalence of eosinophils in BALF had significantly increased within 1 hr of i.p. injection of PAF (10 micrograms/animal) and was maximal after 24 hr. Subcutaneous osmotic mini-pumps were used to administer drugs for 5 days prior to i.p. injection of PAF (10 micrograms/animal) and for the subsequent 24 hr. The percentage increase of eosinophils in BALF, due to PAF, was inhibited in animals treated with dexamethasone, aminophylline, cromoglycate, tranilast or ketotifen, but not in animals treated with oxatomide, azelastine, amlexanox, ibudilast or AA-861. These results suggest that inhibition of pulmonary eosinophilia may be a necessary property of prophylactic anti-asthma drugs and provide indirect evidence favoring a role for PAF in eosinophilia of asthma.

It has been proposed that the actions of platelet activating factor (PAF) could ac count for the development and exacerbation of asthma that can results from allergic or non allergic processes ('1 ). In this context, it has been established that systemic administration of PAF will induce airway hyperreactivity (2) and that prophylactic anti-asthma drugs can prevent development of such airway hyper reactivity (3). More recently, it has been established that inhalation (4) or intratracheal instillation (5) of PAF can also induce a selective accumulation of eosinophils that can be prevented by ketotifen (5,6) and other prophylactic anti-asthma drugs (6). These observations raise the possibility that inhi bition of eosinophil accumulation may be a property common to anti-asthma drugs.
The present study demonstrates the capaci ty of intraperitoneal injection of PAF to effect eosinophil accumulation in the airways and has defined the capacity of prophylactic anti asthma drugs to inhibit this process.
Cell suspensions were concentrated by low speed centrifugation (200xg for 10 min), and the cell pellet was resuspended in 1 ml modified Tyrode. Ten microliters of cell sus pension were diluted in 90 /I of Turk's fluid for total cell counts. Differential cell counts were made from smears fixed in methanol (100%) and stained with Leishman's stain in order to differentiate cell types. A total of ca. 500 cells per smear were counted at 1000 fold magnification.
Time and dose-related increases in bron chial eosinophils: To determine the optimal time for bronchial eosinophil accumulation, guinea pigs were injected with PAF (10 ,ag/ animal in 5 ml of BSA-saline, i.p.); and 1, 4, 8, 16, 24 or 48 hr later, bronchial eosinophil counts were determined.
To determine whether responses to PAF were dose-related, increasing concentrations of PAF (0.1-10 /cg/animal, i.p.) were injected, and bronchial eosinophil counts were deter mined at 24 hr. Injections of BSA-saline alone acted as the control for all experiments.
Inhibition of eosinophil accumulation in pulmonary airways: Drugs were administered to guinea pigs as a subcutaneous infusion over a 6 day period, using osmotic mini pumps (Alzet 2001, 2 ml). Pumps were im planted subcutaneously in the nuchal region under ether anesthesia, 5 days prior to in traperitoneal injection of PAF. Twenty-four hours after injection of PAF, BAL was per formed and eosinophil counts were deter mined.

Results
The time course of eosinophil accumulation in BALF was examined following i.p. injection of 10 ag/animal PA.F. There was an increase in the percentage of eosinophils 1 hr after i.p. injection of PAF or BSA-saline (Fig. 1). How ever, the percentage of eosinophils in BALF of the PAF treated animals gradually in creased and was maximal at 24 hr (Fig. 1), whereas no such increase was observed in the BSA-saline treated animals (Fig. 1).

Intraperitoneal
injection of PAF (0.1-10 ,cg/ animal) induced a dose-related increase in the total number and percentage of eosinophils in BALF at 24 hr (Fig. 2), although no increase in total leukocyte number was observed ( Table 1).
The effect of drug treatment for 5 days prior to exposure to PAF is shown in Table 2. There was a significant decrease in the per centage of eosinophils in BALF from animals treated with dexamethasone, cromoglycate, tranilast or ketotifen. Treatment with equiva lent doses of oxatomide, azelastine, amlex anox, ibudilast or AA-861 failed to diminish eosinophil accumulation. Aminophylline inhi bited eosinophil accumulation when ad ministered at 10 mg/kg/day.

Infiltration
of the bronchial wall and airways by eosinophils is a characteristic of asthma, being evident not only when asthma patients die in status asthmaticus (7), but also death is due to causes unrelated to asthma (8). Bronchial eosinophilia has also been observed in biopsy studies on specimens from asthma patients (9). The association between increased eosinophil counts and severity of asthma is strengthened by the observations that bronchial eosinophil counts increase when there is a late-onset asthmatic reaction following aerosol challenge with the provocative antigen (10) and the presence of a positive correlation between blood eosino phil numbers and increased airway reactivity (11). Major basic protein (MBP) and eosin ophil cationic protein (ECP) are present within eosinophil granules. The concentra tions of such toxic proteins are elevated in bronchial tissue at sites of epithelial damage (8) and in the sputum (12) and blood (13) of asthma patients. MBP is known to damage bronchial epithelium under laboratory con ditions (14) within the concentration range that can be found in asthmatic sputum (15). It has been proposed that damaged epithelium could lead to increased airway reactivity either by exposing underlying nerve endings (16) or by removing the protective effect of an epithelial-derived relaxant factor (17). Recent studies have confirmed that epithelial damaged with MBP can lead to increased contractility of airway smooth muscle in vitro (18).
Despite the importance of eosinophils in the pathology of asthma, the actions of anti asthma drugs on eosinophil function have not been studied extensively. Studies in the rat (19), guinea pig (6) and mouse (20) indicate that drugs such as ketotifen or cromoglycate may achieve inhibition irrespective of the stimulus used to induce eosinophil accumu lation. There are few clinical reports on the effect of anti-asthma drugs on eosinophils in asthma. Treatment with steroids is an effec tive means of suppressing eosinophilia in peripheral blood (21,22), and both ketotifen and theophylline have been reported to diminish eosinophil counts in peripheral blood (23,24). Cromoglycate has been reported to diminish the number of eosinophils recovered from airway lavage fluid in asthma (25). Recently, the activation state of eosinophils in allergic diseases has attracted interest, and it  has been reported that ketotifen treatment de creased the number and proportion of hy podense (light density, 26) eosinophils in the peripheral blood of patients with atopic dermatitis (27).
In the present study, marked inhibition of airway eosinophilia was achieved by dex amethasone, aminophylline, cromoglycate, tranilast and ketotifen. Other drugs tested in this study exhibit a range of properties that could lead to anti-allergic activity including mast cell stabilization and histamine (H1) antagonism (azelastine and oxatomide), leukotriene antagonism (amlexanox and ibudilast) and lipoxygenase inhibition (AA 861). None of these compounds influenced eosinophil accumulation in the airway lumen of guinea pigs, although it has been reported elsewhere that oral administration of ibudilast inhibits pulmonary eosinophilia due to antigen (28).
The capacity of prophylactic anti-asthma drugs to inhibit eosinophilia of the airways due to PAF cannot be associated with the known biological effects of these drugs. An effect of the drugs on platelets might be anticipated, since cromoglycate, ketotifen, aminophylline and steroids inhibit the de velopment of airway hyperreactivity in re sponse to i.v. infusion of PAF, a process known to be platelet-dependent (2), and there have reports of effects of such drugs upon platelet activation (29). However, in contrast to airway hyperreactivity in the guinea pig, PAF-induced eosinophil accumu lation is not subordinate to platelet activation (4). The conclusion that induction of airway hyperreactivity and eosinophil accumulation by PAF are distinct events is reinforced by the evidence that tranilast inhibits eosinophil ac cumulation but not airway hyperreactivity (30), whereas ibudilast inhibits PAF-induced hyperreactivity (30) but not eosinophil ac cumulation.
The findings of the present study are con sistent with the concept that PAF formation may determine accumulation of eosinophils in asthma. These results suggest that inhibition of eosinophil accumulation in the airways is an essential component of the pharmacolo gical action of prophylactic anti-asthma drugs and that the guinea pig treated with PAF may be an appropriate model for prophylactic anti asthma drug evaluation. airway hyperreactivity that follows exposure to platelet activating factor (PAF  23, 187-194 (1988)