Bronchial Challenge with Adenosine Causes the Release of Serum Neutrophil Chemotactic Factor in Asthma 1- 3

ALBERT G. DRIVER, CYNTHIA A. KUKOLY, W. JAMES METZGER, and S. JAMAL MUSTAFA

Introduction

Asthmatics develop bronchoconstriction when they inhale nebulized solutions of adenosine. This response to adenosine has been demonstrated in allergic and non allergic individuals (1). Adenosine is 17-fold less potent than methacholine and approximately 4-fold lesspotent than histamine in this regard (2, 3). Histamine is 20-fold more potent than the related nucleotide adenosine 5'-monophosphate (AMP) as a bronchoconstricting substance (4). The mechanism of adenosineinduced bronchoconstriction is unknown. Most of the evidence that has been accumulated thus far suggests that adenosine causes bronchospasm by stimulating or enhancing the release of mast cell-derived mediators of bronchoconstriction. The effects of adenosine are inhibited by pretreatment with HI antihistamines, cromolyn sodium, and nedocromil (4-7). Pretreatment with ipratropium bromide has no effect on inhibiting the response to adenosine (2). Theophylline also has been found to inhibit adenosine-induced bronchoconstriction (8). Theophylline is an adenosine receptor antagonist, and this property of the drug may contribute to its therapeutic effect in asthma. A related xanthine derivative, enprofylline, has negligible adenosine antagonistic prop.. erties and also has bronchodilating effects in allergic asthma (9). Adenosine has been shown to potentiate the release of mediators from human lung mast cells,human basophils, and rat mast cells in vitro (10-13). Enhanced release of histamine into peripheral- blood was found in a recent study of AMP-induced bronchoconstriction in 10 atopic nonasthmatic subjects (14). The degreeof elevation of plasma histamine was only half that observed when the same subjects were challenged with antigen. However, another study involving eight asthmatic patients did not find evidence of neutrophil chemotactic activity (NCA) in the serum after adenosine challenge (15). High molecular weight NCA has been 1002

SUMMARY In order to Investigate the mechanism of adenosine-Induced bronchoconstrletlon In asthma, serum neutrophil chemotactic activity (NCA)was measured In normal Individuals and patients with asthma before and 5 min after bronchoprovocatlon testing with adenosine. Challenge testing was terminated when the FEV1 fell by 20% or a concentration of 10 mg/ml was reached. Participants were separated Into three groups: six asthmatics hyperresponslve to adenosine (Group 1), seven asthmatics hyperresponslve to histamine but not adenosine (Group 2), and six normal subjects (Group 3). The mean percentage Increase In NCA was 84% for Group 1 (p < 0.001),29% for Group 2 (p < 0.05), and only 13% for Group 3. No significant Increase In NCA was observed after histamine challenge In seven Individuals with asthma derived from Groups 1 and 2. Four patients from Group 1 were rechallenged after treatment with therapeutic doses of oral theophylline. Theophylline therapy was associated with a significant attenuation of the Increase In NCA at the concentration of adenosine which caused a 20% decrease In FEV1 before treatment (18% versus 84%, p < 0.01). The concentration of adenosine which caused a 20% drop In FEV1 was Increased at least twofold for each of the four patients. Analysis of NCA by gel filtration chromatography demonstrated an Increase In a high molecular weight neutrophil chemotactic factor In the serum of two Group 1 patients after adenosine challenge. Release of a high molecular weight neutrophil chemotactic factor Is consistent with a mast cell source for Inflammatory mediators In adenosine-Induced bronchoconstrletlon. The therapeutic effects of theophylline, a potent adenosine antagonist, In asthma may therefore occur In part through the Inhibition of this process. AM REV RESPIR DIS 1991; 143:1002-1007

considered a marker for the release of mediators by mast cellsduring early and late asthmatic reactions (16-18). In order to further investigate the mechanism of adenosine-induced bronchoconstriction and to resolve these conflicting observations, serum NCA was measured before and after adenosine challenge in asthmatic patients and normal subjects. The effect of theophylline on adenosineinduced bronchoconstriction also was examined. Methods Subject Selection Thirteen nonsmoking asthmatic patients 20 to 60 yr of age and six normal subjects aged 25 to 65 were studied after giving informed consent. The experimental protocol was approved by the Institutional Review Committee of the Pitt County Memorial Hospital and the East Carolina University School of Medicine. All participants refrained from using xanthine-containing drinks for 6 h before testing. Asthmatic patients used only inhaled beta-agonists and inhaled or oral corticosteroids during the time of the study. Inhaled beta-agonists were not used less than 61) prior to adenosine challenges.

Aerosol Bronchoprovocation Spirometry was performed using a pneumotach system(Spinnaker Spirometer;Cybermedics, Inc, Colorado Springs, CO). Baselinelung function was measured after the inhalation of nebulized saline and two measurements were made to ensure reproducibility. Spirometric results for FEV 1 were compared with published predicted values (19). Histamine and adenosine solutions were prepared by dissolving histamine dihydrochloride and adenosine-free base (Sigma Chemicals, St. Louis, MO) in 0.9070 saline. Prior to nebulization, the solutions were sterilized by filtration through a 0.2 J..L Gelman Acrodisc (Receivedin originalform August 29, 1990and in revised form December 14, 1990) 1 From the Departments of Medicine and Pharmacology,East Carolina UniversitySchool of Medicine, Greenville, North Carolina. 2 Supported by Grant HL-36394 from the National Institutes of Health, the Trudeau Scholar Award from the American Lung Association, and a grant from the American Lung Association of North Carolina. 3 Correspondence and requests for reprints should be addressed to Dr. Albert G. Driver,Department of Medicine,ECD School of Medicine,Greenville, NC 27858-4354.

BRONCHIAL ADENOSINE CHALLENGE AND SERUM NCA IN ASTHMA

filter (Gelman Sciences, Ann Arbor, MI). Adenosine solutions wereprepared in the following concentrations: 0.03, 0.07, 0.155,0.31, 0.65, 1.25, 2.5, 5.0, and 10.0 mg/ml. Histamine solutions wereused in the following concentrations: 0.03, 0.06, 0.12,0.25, 1.0,2.5,5.0, and 10.0 mg/ml. Histamine and adenosine solutions were nebulized with Airlife disposable nebulizers (American Pharmaseal Co., Valencia, CAl. Only nebulizers that delivered 0.3 ± 0.03 mg/min at air flow rates of 6 to 7 L/min were used. The participants inhaled nebulized solutions by tidal breathing with 16breaths taken over 1 min. Nose clips were worn during inhalation and during pulmonary function testing. Inhalational challenges were terminated when a 20070 fall in FEV 1 from baseline occurred (PC 2 0 ) or when the highest concentration of histamine or adenosine was reached. Patients were divided into two groups depending on the response of inhaled adenosine or histamine. Group 1 consisted of 6 patients who werehyperresponsive to adenosine (defined by a 20% decrease in FEV 1 at any concentration up to and including 10mg/ml). Bronchial response to inhaled histamine was not mea.suredin three members of this group. The second group (Group 2), consisting of seven patients, werehyperresponsive to histamine (PC 20 < 10 mg/ml) but not adenosine. Group 3 consisted of six subjects with no respiratory symptoms and normal pulmonary function tests. These subjects were matched for age and sex with the six patients in Group 1.

Neutrophil Chemotaxis Blood for NCA was drawn before each challenge, 5 and 30 min after the highest concentration of either histamine or adenosine was inhaled. Serum was obtained from the blood and frozen at -70° C until analysis. In a separate series of experiments, four asthmatic subjects from Group 1 were treated with theophylline at the maximum tolerated dose for each individual. Bronchial challenge with adenosine was repeated after 2 days of therapy. Blood samples were drawn before challenge and 5 min after inhalation of the dose of adenosine that had previously caused a 20% decrease in FEV l ' Inhalation challenge was then continued until the FEV 1 fellby 20% or the 10 mg/ml solution had been inhaled. A third sample of blood was obtained 5 min after inhalation of the highest concentration of adenosine. A portion of the serum from this experiment was saved for determination of the serum theophylline concentration. Serum NCA was evaluated using a microchemotaxis chamber method (NeuroProbe, Inc., Cabin John, MD) (20). Twenty-six microliters of serum diluted 1:20with Hanks' buffered saline (HBSS) was placed in the bottom wellof a 48-well chemotaxis chamber and covered with a 3-1l pore size polyvinylpyrrolidone (PVP)-free polycarbonate membrane. Neutrophils were collected from normal volunteers using dextran sedimentation. Neutrophils wereobtained in 80% purity. The cells

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were suspended in HBSS and added to the 50-Ill top well (40,000 cells per well). Triplicate determinations were made for each experimental condition. The chambers were incubated for 40 min in humidified air at 37° C. Neutrophil migration was measured after fixing and staining with Diff-Quik" (American Scientific,McGaw Park, IL). Cells were counted under high-power magnification (x 450) by light microscopy. Cells were counted in 10 randomly selected fields by a blind analysis. The results were expressed as the number of neutrophils that had traversed the filter in 10 high-power fields (HPF). The positive and negative controls were a 1:10dilution of zymosan activated serum and HBSS, respectively. Molecular weight determination of NCA was made on samples obtained from three Group 1patients before and 5 min after adenosine challenge. The molecular weight of NCA was also determined in prechallenge serum from three normal subjects. Gel filtration was performed on columns of Sephadex o-zoo (1.5 X 90 cm; Pharmacia Fine Chemicals, Piscataway, NJ) as previously described (16-18). The column was standardized using blue dextran, cytochrome C, and phenol red as molecular weight standards. Samples were dialyzed overnight (Spectra/Por 6 membranes, 3,500 molecular weight cut off; Spectrum Medical Industries, Inc., Los Angeles, CAl prior to chromatography. The columns were developed at 4 to 6 mllh and fractions of 2 ml were collected. Alternate undiluted samples weretested in duplicate for NCA. Preand postchallenge fractions were paired for direct comparison in each assay. Complete curves of neutrophil counts for the entire range of molecular weights were constructed by subtracting background counts obtained from the negative controls and expressing the results as a percentage of the positive controls for each assay. Heat stability of the high molecular weight fractions were determined by heating undiluted samples at 56° C for 1 h and assaying for NCA.

Statistical Analysis Results were compared using Student's t test and analysis of variance. A value of p < 0.05 was considered significant. Results

Subject Characteristics Six asthmatics were found to be hyperresponsive to adenosine (Group 1). Four of these were being treated with inhaled

beta-agonists at the time of the study. 1\vo of these patients also were treated with prednisone and one patient also was treated with inhaled flunisolide.1\vowere not taking any medication. Seven patients with asthma were found to be hyperresponsive to histamine but not adenosine (Group 2). Three of this group were being treated with inhaled beta agonists. The control group of normal subjects with no history of respiratory symptoms comprised Group 3. Baseline lung function and patient characteristics are summarized in table 1. Subjects in Group 1had poorer mean lung function, whether measured by either FEV 1 as a percentage of predicted values or by the FEV1/FVC ratio. The geometric mean PC 2 0 for histamine in 3 of the patients in this group was 0.43 mg/ml, whereas the geometric mean PC20 for adenosine was 1.4 ± 1.5mg/ml for the entire group. Not all members of Group 1 were challenged with histamine because baseline lung function was abnormal in all but one. Patients in Group 2 had an intermediate level of lung function, whereas subjects in Group 3 had normal lung function. Those in Group 2 had a reduction in FEV 1 varying from 0 to 16070 of baseline (mean, 6070) after inhalation of 10 mg/ml of adenosine. The geometric mean PC 20 for histamine in this group was 3.4 ± 0.5 mg/ml. No subject in Group 3 had a significant reduction in FEV 1 with adenosine inhalation. Bronchial hyperresponsiveness to histamine wasnot measured in these subjects as they had no history of respiratory symptoms and their pulmonary function tests were normal.

Neutrophil Chemotaxis Results of neutrophil chemotaxis assays are summarized in figure 1. Serum NCA increased from a mean of 284 ± 43 cells/10 HPF before adenosine challenge to 521 ± 53 cells/10 HPF 5 min after inhalation of the highest tolerated concentration of adenosine. This difference was significant at p ~ 0.001.Serum NCA activity was reduced almost to baseline 30 min after challenge. The increase in

TABLE 1 CHARACTERISTICS OF SUBJECTS CHALLENGED WITH ADENOSINE*

Group 1 Group 2 Group 3

N

Age (yr)

Sex

6 7 6

39 ± 5 38 ± 3 38 ± 6

5 male 6 male 5 male

• Results represent the mean ± standard error.

FEV1

%

Predicted

80 ± 9 95 ± 4 104 ± 2

FEV1/FVC 0.69 ± 0.04 0.78 ± 0.03 0.82 ± 0.03

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DRIVER, KUKOLY, METZGER, AND MUSTAFA

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Bronchial challenge with adenosine causes the release of serum neutrophil chemotactic factor in asthma.

In order to investigate the mechanism of adenosine-induced bronchoconstriction in asthma, serum neutrophil chemotactic activity (NCA) was measured in ...
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