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The Role of Free Radicals in Airway Obstruction in Asthmatic Patients
The Role of Free Radicals in Airway Obstruction in Asthmatic Patients· Hiroshi Kanazawa, M.D.; Naotsugu Kurihara, M.D., F.C.C.P.; Kazuto Hirata, M.D.; and Tadanao Takeda, M.D., F.C.C.R Neutrophils from asthmatic patients seem to be in an activated state. This study demonstrated that superoxide radical (Os- ) production was enhanced in asthmatic patients compared with healthy control subjects. Production of (Os- ) also increased with progression of disease and with disease duration. In addition, (Os- ) release was inversely correlated with FEV. and midftows and was greater in neutrophils from patients with exacerbated disease than in those from
patients with stable disease. These findings suggest that oxygen metabolites may playa direct or indirect role in the modulation of airway in8ammation. (Chest 1991; 100:1319-22)
hyperresponsiveness is a feature character-
complement receptor expression5-7 and increased activity of the 5-lipooxygenase pathway. 8,9 An inference from the foregoing is that neutrophils may act by several different mechanisms in bronchial asthma. They produce mediators that can induce bronchoconstriction, such as platelet activating factor,1O prostaglandins,11 and leukotrienes. Neutrophils also produce superoxide anion and other oxygen free radicals that can damage lung tissue. 12-14 The purpose of this study was to ascertain whether neutrophilderived active oxygen radicals are one ofthe important causative factors responsible for airway inflammation and obstructive reactions.
~irway
1"1 istic of asthma. 1However, its precise cause is yet
to be defined despite many clinical investigations. According to the present understanding of asthma, cells of different kinds are involved in the occurrence of symptoms and the persistence of the disease state. The essential role of mast cells and possibly basophils in IgE-mediated responses was suggested a long time ago. Ample data published recently raised the possibility that airway inflammation due to neutrophil infiltration may lead to hyperresponsiveness. 2 Neutrophils are increased in number in human bronchoalveolar lavage fluid after inhalation challenge with antigen. 3 ,4 In addition, neutrophils are likely to be activated in asthmatic patients, as suggested by the increased *From the First Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. Manuscript received December 7; revision accepted March 4. Reprint requests: Dr. Kanazawa, 1-5-7 Asahi-machi, Abenoku, 1st Department of Internal Medicine, Osaka City University Medical School, Osaka, japan 545
DMSO=dimethyl sulfoxide; fMLP= n-fonnyl methionylleucyl phenylalanine; HBSS = Hanks' balanced salt solution; PMA = phorbol myristate acetate; SOD = superoxide dismutase.
SUBJECTS AND METHODS
Subjects The subjects who participated in this study were 11 patients with bronchial asthma, ranging in age from 21 to 69 years (mean, 43.5 years) {Table 1), and five healthy control subjects, ranging in age from 27 to 70 years (mean, 45.0 years). The two groups were matched by age and sex. All asthmatic subjects were clinically stable nonsmokers who had never experienced exacerbation of symptoms
Table I-Clinical CharacteriBtica a/the Study Group Pulmonary Function Test Subject
Age, yr
Sex
Atopic or Nonatopic
1 2 3 4 5 6 7 8 9 10 11
24 59 69 39 53 39 57 21 32 37 49
F F F F M F M M F M M
Nonatopic Nonatopic Nonatopic Atopic Nonatopic Atopic Atopic Atopic Nonatopic Atopic Atopic
~.esultst
Medication*
%TLC
FEVI.M>
RVlfLC
T,B T,B,A T,B,A T,B T,B T,B T,B,A B
lOB.8 84.4 100.0 125.4 114.5 85.0 107.1 113.3 94.8 98.0 103.2
82.5 73.9 66.9 84.1 78.9 64.2 46.9 74.1 82.4 80.5 80.0
25.1 34.3 42.2 37.8 25.0 36.5 40.0 17.9 21.6 25.0 33.5
B B
*A = anticholinergic; B = beta-stimulant; T = theophylline. tTLC = total lung capacity; RV = residual volume; TLC = total lung capacity. CHEST I 100 I 5 I NOVEMBER, 1991
1319
and had no signs suggestive of respiratory infection for at least one month before the stud~ A diagnosis of asthma had already been established for each patient based on symptoms and the reversibility ofairftow limitation by bronchodilators. . Methacholine inhalation challenge testing was done for all subjects. All challenge testing was done at approximately 1:00 PM to eliminate diurnal variation. After baseline spirometry and inhalation ofdiluent to establish stability of FEVI' each subject was instructed to take slow inspirations of each concentration of methacholine. Spirometry was performed 3 min after each set of inhalations. All asthmatic patients in this study demonstrated bronchial hyperreactivity to methacholine to various degrees (data not shown). The patients had been receiving oral or inhaled theophylline, betastimulants, and anticholinergic drugs. CeU Preparation
Human neutrophils were isolated from heparinized venous blood through the sequential application of Ficoll-Hypaque centrifugation l5 and 3 percent dextran sedimentation. Contaminating erythrocytes were lysed by exposure to hypotonic saline. The isolated neutrophils were washed and resuspended in Hanks' balanced salt solution (HBSS) at a concentration of 1()8 cells per milliliter. This procedure yielded cells with a viability of not less than 95 percent by trypan blue dye exclusion and purity of not less than 98 percent by May-GrUnwald staining. Superoxide Radical Generation
Generation of 0 1 - was assessed by the superoxide dismutase (SOD) inhibitable reduction of ferricytocbrome C.IS Neutrophils (1 x 1()8/ml) were suspended in HBSS and incubated at 3rc for 10 min. Cytochrome C and either phorbol myristate acetate (PMA) (final concentration, 10 nglml) or n-formyl methionylleucyl phenylalanine (fMLP) (final concentration, 10- 7 mollL) were added to the cell suspension. The PMA and fMLP were dissolved in dimethylsulfoxide (DMSO). Addition of DMSO alone was ineffective. The reaction was terminated after 10 min by cooling the sample to OOC and centrifuging it at 2,000 rpm for 10 min. The supernatant was then decanted, and the amount of reduced cytochrome C produced was assessed by reading at 550 nm in a spectrophotometer. All reactants plus SOD were used in the control experiment. The amount of O.- produced was calculated, as well as the amount of cytochrome C reduced in each tube, using an extinction coefficient of 2.95 x I~M-lecm-I"7 Statistical Evaluation
Generation of O 2 - was determined in triplicate for each patient at the saturating dose of fMLP or PMA. The arithmetic means obtained for the asthmatic patients and the healthy control subjects were compiled, and the significance was evaluated with Student's t test. The possible correlations of O2 - production to disease duration and pulmonary function were analyzed by a linear regression method. RESULTS
The PMA- or fMLP-stimulated production of O2 by neutrophils was significantly greater in the asthTable 2- Supercnide Anion Production in ABthmatic Patients and Normal Control Subjecta
Superoxide Anion Production (nmollmin) Stimulant
Controls (n = 5)
Asthmatics (n = 11)
PMA (10 nglml) fMLP (10- 7 mollL)
1.25±0.48 0.76±0.24
2.14±0.70* 1.49±0.25*
*p
1320
c: 'E 4.0
~
"" (5 E c:
'-"
"'C
••
3.0
•
Q)
0 :J "'C Q) L.
u
2.0
Q)
E
•
o 1.0 L.
.t:.
o o
~
>.
o
o
10
• r=O.6 p
20
disease duration (years)
FIGURE 1. Correlation between disease duration and superoxide anion production after stimulation with PMA.
matic patients than in the normal control subjects (Table 2). Comparison between the two asthmatic subgroups, continuous inhalers of bronchodilators (who had attacks frequently) and intermittent inhalers, showed that O2 - production was apt to be greater in the former subgroup (02 - release; 2.57 ± 0.70 vs 1.70 ± 0.30 nmoVml). There was a positive correlation between disease duration and O2 - production: the patients with a long disease duration tended to have a high level of O2 - production (Fig 1). In addition, 2 production had a negative correlation to FEVI and V25, which represent pulmonary function: O2 - production was apt to be active in the patients showing obstructive patterns of responses to pulmonary function tests (Fig 2). In five asthmatic patients, 2 production could be studied in both episodic and stable phases. The results obtained indicate that 2 production was enhanced during an asthmatic attack (Fig 3).
°
° °
DISCUSSION
This study revealed the O2 - production by peripheral blood neutrophils was distinctly enhanced in asthmatic patients compared with healthy control subjects. A majority of our patients were receiving theophylline and beta-stimulatants. These substances, which are known to increase the intracellular concentration of cyclic adenosine monophosphate,18 must have inhibited O2 - generation by peripheral blood neutrophils, that is, the O2 - production was most probably underestimated in our patients. Whichever the case may be, the O2 - production was positively correlated with disease severity and duration. Free Radicals in AJrway Obstruction in Asthma (Kanazawa et 8/)
......... .~
E .......
'£ 4.0
4.0
E
oS 3.0
E
.......
•
•
(5
(5
."
..s 3.0 ."
•
Q)
u
:J ."
f 2.0
0 Q)
•
E
oL 1.0 0
,
~
~
u
¥O
50
,
60
,
0 :J
• • .1 •
,
."
70 80 FEV 1.0"
•
f 2.0
0
•
•
Q)
r=-0.7 P
.s:.
u
•
Q)
•
•
•
E
,
90 (,,)
•
• •
oE 1.0
••
s:L
0
0
~ 0
0
1.0
V25
r=-0.78 P
FIGURE 2. Correlation between pulmonary function test results and superoxide anion production after stimulation with PMA.
Furthermore, O2 - production had a negative correlation with FEV. and V25. The correlation with the latter parameter, which represents the pathologic state of the minute respiratory tracts, was especially strong. This fact furnishes a basis for the hypothesis that O2 plays some role in the development of bronchospasm. The enhancement of O2 - production during an asthmatic attack seems to support this hypothesis. Neijens et al l9 also found that leukocytes from asthmatic children generated more O2 - than polymorphonuclear leukocytes from control subjects. However, the mechanism of O2 - generation by neutrophils in asthma still remains obscure. The potential importance of neutrophils has been demonstrated in animals by the fact that neutrophil depletion leads to abrogated bronchial hyperreactivit}: m There is considerable evidence to substantiate that an inHammatory infiltrate is essential for the development of human ailway hyperresponsiveness. 21 ,22 P
Superoxide production (nmol/min)
4.
3.0 2.0 e subject No. 1 o No.2
1.0
1
)(
No.3
[•
No. 10 No. II
D
0.....- -....- - -....- asthmatic attack
stable
state
FIGURE 3. Measurement of superoxide anion production after stimulation with PMA in both episodic and stable phases.
Although it has been suggested that the production of cyclooxygenation of arachidonic acid is a primary pathogenetic factor, O2 - generation that causes a local tissue injury might also be contributory. In conclusion, the present observations endorse the correlation between disease severity and oxygen radical production by neutrophils in asthmatic patients and suggest that oxygen metabolites may playa direct or indirect role in the modulation of airway inHammation. REFERENCES
1 Juniper ET, Firth PA, Hargreave FE. Airway responsiveness to histamine and methacholine: relationship to minimum treatment control symptoms of asthma. Thorax 1981; 36:575-79 2 Chung leF. Role of inOammation in the hyperreactivity of the airways in asthma. Thorax 1986; 41:657-62 3 Metzger WJ, Richerson HB, Worden Ie, Monick M, Hunningbake GW Bronchoalveolar lavage of allergic patients following allergen bronchoprovocation. Chest 1986; 89:477-83 4 Metzger WJ, Zavala D, Richerson HB. Local allergen cballenge and bronchoalveolar lavage of allergic asthmatic lungs. Am Rev Respir Dis 1987; 135:433-40 5 Gin ~ Kay AB. The effect of corticosteroids on monocyte and neutrophil activation in bronchial asthma. J Allergy Clio Immunoll985; 76:675-82 6 Gin ~ Shaw 8J, Kay AB. Airways reversibility after predonisolone therapy in chronic asthma is associated with alteration in leukocyte function. Am Rev Respir Dis 1985; 132:1199-1203 7 Ann J~ Walport JJ, Loe TH. Expression of complement receptors type 1 (CRl) and type 3 (CR3) on circulating granulocytes in experimentally provoked asthma. J Allergy Clin Immunol 1989; 83:649-55 8 WangSR, YangCM, Wang SSM, HanSH,ChiangBN. Enhancement of A 23187-induced production of the slow-reacting substance on peripheral leukocytes from subjects with asthma. J Allergy Clin Immunoll986; 77:465-71 9 Mita H, Yui Y, Taniguchi N, Yasuda H, Shida T. Increased activity of 5-lipooxygenase in polymorphonuclear leukocytes from asthmatic patients. Life Sci 1985; 37:W1-14 10 80rgeat ~ Samuelsson B. Arachidonic acid metabolism in polymorphonuclear leukocytes: effects of ionophone A 23187. Proc Natl Acad Sci USA 1979; 76:2148-52 11 Goldstein 1M, Malmsten CL, Samuelsson B, Weissman G. Prostaglandins, thromboxanes, and polymorphonuclear leukoCHEST I 100 I 5 I NOVEMBER, 1991
1321
cytes. Inflammation 1977; 2:309-17 12 Babior BM. Oxygen-dependent microbial killing by phagocytes. N Engl J Med 1978; 298:721-25 13 Shasby DM, Vandenthuysen KM, Tate RM, Shasby SS, McMurtry I, Repine JE. Granulocytes mediate acute edematous lung injury in rabbits and in isolated rabbit lungs perfused with PMA: role ofoxygen radicals. Am Rev Respir Dis 1982; 125:44347 14 Petrone WF, English DK, Wong K, McCord JM. Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc Natl Acad Sci USA 1980; 1159-63 15 Boyum A. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at Ig. Scand J Clin Lab Invest 1968; 21(suppl 97):77-89 16 McCord JM, Fredorich I. Superoxide dismutase. J BioI Chern 1969; 224:6049-55 17 Massey ~ The microestimation of succinate and the extinction
1322
18
19
20
21
22
coefficient of cytochrome C. Biochem Biophys Acta 1969; 34:225-56 Marone G, Thomas LL, Lichtenstein LM. The role of agonists that active adenylate cyclase in the control of cAHP metabolism and enzyme release by human polymorphonuclear leukocytes. J Immunol 1980; 125:2277-83 Neijens HJ, Raateep RE, Degenhart J, Duiverman EJ, Kerrebijn KF. Altered leukocyte response in relation to the basic abnormality in children with asthma and bronchial hyperresponsiveness. Am Rev Respir Dis 1984; 130:744-47 O'Byrne PM, Walters EH, Gold BD. Neutrophil depletion inhibits airway hyperresponsiveness induced by ozone exposure. Am Rev Respir Dis 1984; 130:214-19 Holtzman MJ, Fabbri LM, O'Byrne PM, Gold BD, Aizawa H, Walters EH, et ale Importance of airway inflammation for hyperresponsiveness induced by ozone. Am Rev Respir Dis 1983; 127:686-90 Seltzer J, Bigby BG, Stulbarg M. Ozone-induced change in bronchial reactivity to methacholine and airway inflammation in humans. J Appl Physiol 1986; 60: 1321
Free Radicals in Airway Obstruction in Asthma (KsnszsW8 et 81)
patients with stable disease. These findings suggest that oxygen metabolites may playa direct or indirect role in the modulation of airway in8ammation. (Chest 1991; 100:1319-22)
hyperresponsiveness is a feature character-
complement receptor expression5-7 and increased activity of the 5-lipooxygenase pathway. 8,9 An inference from the foregoing is that neutrophils may act by several different mechanisms in bronchial asthma. They produce mediators that can induce bronchoconstriction, such as platelet activating factor,1O prostaglandins,11 and leukotrienes. Neutrophils also produce superoxide anion and other oxygen free radicals that can damage lung tissue. 12-14 The purpose of this study was to ascertain whether neutrophilderived active oxygen radicals are one ofthe important causative factors responsible for airway inflammation and obstructive reactions.
~irway
1"1 istic of asthma. 1However, its precise cause is yet
to be defined despite many clinical investigations. According to the present understanding of asthma, cells of different kinds are involved in the occurrence of symptoms and the persistence of the disease state. The essential role of mast cells and possibly basophils in IgE-mediated responses was suggested a long time ago. Ample data published recently raised the possibility that airway inflammation due to neutrophil infiltration may lead to hyperresponsiveness. 2 Neutrophils are increased in number in human bronchoalveolar lavage fluid after inhalation challenge with antigen. 3 ,4 In addition, neutrophils are likely to be activated in asthmatic patients, as suggested by the increased *From the First Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. Manuscript received December 7; revision accepted March 4. Reprint requests: Dr. Kanazawa, 1-5-7 Asahi-machi, Abenoku, 1st Department of Internal Medicine, Osaka City University Medical School, Osaka, japan 545
DMSO=dimethyl sulfoxide; fMLP= n-fonnyl methionylleucyl phenylalanine; HBSS = Hanks' balanced salt solution; PMA = phorbol myristate acetate; SOD = superoxide dismutase.
SUBJECTS AND METHODS
Subjects The subjects who participated in this study were 11 patients with bronchial asthma, ranging in age from 21 to 69 years (mean, 43.5 years) {Table 1), and five healthy control subjects, ranging in age from 27 to 70 years (mean, 45.0 years). The two groups were matched by age and sex. All asthmatic subjects were clinically stable nonsmokers who had never experienced exacerbation of symptoms
Table I-Clinical CharacteriBtica a/the Study Group Pulmonary Function Test Subject
Age, yr
Sex
Atopic or Nonatopic
1 2 3 4 5 6 7 8 9 10 11
24 59 69 39 53 39 57 21 32 37 49
F F F F M F M M F M M
Nonatopic Nonatopic Nonatopic Atopic Nonatopic Atopic Atopic Atopic Nonatopic Atopic Atopic
~.esultst
Medication*
%TLC
FEVI.M>
RVlfLC
T,B T,B,A T,B,A T,B T,B T,B T,B,A B
lOB.8 84.4 100.0 125.4 114.5 85.0 107.1 113.3 94.8 98.0 103.2
82.5 73.9 66.9 84.1 78.9 64.2 46.9 74.1 82.4 80.5 80.0
25.1 34.3 42.2 37.8 25.0 36.5 40.0 17.9 21.6 25.0 33.5
B B
*A = anticholinergic; B = beta-stimulant; T = theophylline. tTLC = total lung capacity; RV = residual volume; TLC = total lung capacity. CHEST I 100 I 5 I NOVEMBER, 1991
1319
and had no signs suggestive of respiratory infection for at least one month before the stud~ A diagnosis of asthma had already been established for each patient based on symptoms and the reversibility ofairftow limitation by bronchodilators. . Methacholine inhalation challenge testing was done for all subjects. All challenge testing was done at approximately 1:00 PM to eliminate diurnal variation. After baseline spirometry and inhalation ofdiluent to establish stability of FEVI' each subject was instructed to take slow inspirations of each concentration of methacholine. Spirometry was performed 3 min after each set of inhalations. All asthmatic patients in this study demonstrated bronchial hyperreactivity to methacholine to various degrees (data not shown). The patients had been receiving oral or inhaled theophylline, betastimulants, and anticholinergic drugs. CeU Preparation
Human neutrophils were isolated from heparinized venous blood through the sequential application of Ficoll-Hypaque centrifugation l5 and 3 percent dextran sedimentation. Contaminating erythrocytes were lysed by exposure to hypotonic saline. The isolated neutrophils were washed and resuspended in Hanks' balanced salt solution (HBSS) at a concentration of 1()8 cells per milliliter. This procedure yielded cells with a viability of not less than 95 percent by trypan blue dye exclusion and purity of not less than 98 percent by May-GrUnwald staining. Superoxide Radical Generation
Generation of 0 1 - was assessed by the superoxide dismutase (SOD) inhibitable reduction of ferricytocbrome C.IS Neutrophils (1 x 1()8/ml) were suspended in HBSS and incubated at 3rc for 10 min. Cytochrome C and either phorbol myristate acetate (PMA) (final concentration, 10 nglml) or n-formyl methionylleucyl phenylalanine (fMLP) (final concentration, 10- 7 mollL) were added to the cell suspension. The PMA and fMLP were dissolved in dimethylsulfoxide (DMSO). Addition of DMSO alone was ineffective. The reaction was terminated after 10 min by cooling the sample to OOC and centrifuging it at 2,000 rpm for 10 min. The supernatant was then decanted, and the amount of reduced cytochrome C produced was assessed by reading at 550 nm in a spectrophotometer. All reactants plus SOD were used in the control experiment. The amount of O.- produced was calculated, as well as the amount of cytochrome C reduced in each tube, using an extinction coefficient of 2.95 x I~M-lecm-I"7 Statistical Evaluation
Generation of O 2 - was determined in triplicate for each patient at the saturating dose of fMLP or PMA. The arithmetic means obtained for the asthmatic patients and the healthy control subjects were compiled, and the significance was evaluated with Student's t test. The possible correlations of O2 - production to disease duration and pulmonary function were analyzed by a linear regression method. RESULTS
The PMA- or fMLP-stimulated production of O2 by neutrophils was significantly greater in the asthTable 2- Supercnide Anion Production in ABthmatic Patients and Normal Control Subjecta
Superoxide Anion Production (nmollmin) Stimulant
Controls (n = 5)
Asthmatics (n = 11)
PMA (10 nglml) fMLP (10- 7 mollL)
1.25±0.48 0.76±0.24
2.14±0.70* 1.49±0.25*
*p
1320
c: 'E 4.0
~
"" (5 E c:
'-"
"'C
••
3.0
•
Q)
0 :J "'C Q) L.
u
2.0
Q)
E
•
o 1.0 L.
.t:.
o o
~
>.
o
o
10
• r=O.6 p
20
disease duration (years)
FIGURE 1. Correlation between disease duration and superoxide anion production after stimulation with PMA.
matic patients than in the normal control subjects (Table 2). Comparison between the two asthmatic subgroups, continuous inhalers of bronchodilators (who had attacks frequently) and intermittent inhalers, showed that O2 - production was apt to be greater in the former subgroup (02 - release; 2.57 ± 0.70 vs 1.70 ± 0.30 nmoVml). There was a positive correlation between disease duration and O2 - production: the patients with a long disease duration tended to have a high level of O2 - production (Fig 1). In addition, 2 production had a negative correlation to FEVI and V25, which represent pulmonary function: O2 - production was apt to be active in the patients showing obstructive patterns of responses to pulmonary function tests (Fig 2). In five asthmatic patients, 2 production could be studied in both episodic and stable phases. The results obtained indicate that 2 production was enhanced during an asthmatic attack (Fig 3).
°
° °
DISCUSSION
This study revealed the O2 - production by peripheral blood neutrophils was distinctly enhanced in asthmatic patients compared with healthy control subjects. A majority of our patients were receiving theophylline and beta-stimulatants. These substances, which are known to increase the intracellular concentration of cyclic adenosine monophosphate,18 must have inhibited O2 - generation by peripheral blood neutrophils, that is, the O2 - production was most probably underestimated in our patients. Whichever the case may be, the O2 - production was positively correlated with disease severity and duration. Free Radicals in AJrway Obstruction in Asthma (Kanazawa et 8/)
......... .~
E .......
'£ 4.0
4.0
E
oS 3.0
E
.......
•
•
(5
(5
."
..s 3.0 ."
•
Q)
u
:J ."
f 2.0
0 Q)
•
E
oL 1.0 0
,
~
~
u
¥O
50
,
60
,
0 :J
• • .1 •
,
."
70 80 FEV 1.0"
•
f 2.0
0
•
•
Q)
r=-0.7 P
.s:.
u
•
Q)
•
•
•
E
,
90 (,,)
•
• •
oE 1.0
••
s:L
0
0
~ 0
0
1.0
V25
r=-0.78 P
FIGURE 2. Correlation between pulmonary function test results and superoxide anion production after stimulation with PMA.
Furthermore, O2 - production had a negative correlation with FEV. and V25. The correlation with the latter parameter, which represents the pathologic state of the minute respiratory tracts, was especially strong. This fact furnishes a basis for the hypothesis that O2 plays some role in the development of bronchospasm. The enhancement of O2 - production during an asthmatic attack seems to support this hypothesis. Neijens et al l9 also found that leukocytes from asthmatic children generated more O2 - than polymorphonuclear leukocytes from control subjects. However, the mechanism of O2 - generation by neutrophils in asthma still remains obscure. The potential importance of neutrophils has been demonstrated in animals by the fact that neutrophil depletion leads to abrogated bronchial hyperreactivit}: m There is considerable evidence to substantiate that an inHammatory infiltrate is essential for the development of human ailway hyperresponsiveness. 21 ,22 P
Superoxide production (nmol/min)
4.
3.0 2.0 e subject No. 1 o No.2
1.0
1
)(
No.3
[•
No. 10 No. II
D
0.....- -....- - -....- asthmatic attack
stable
state
FIGURE 3. Measurement of superoxide anion production after stimulation with PMA in both episodic and stable phases.
Although it has been suggested that the production of cyclooxygenation of arachidonic acid is a primary pathogenetic factor, O2 - generation that causes a local tissue injury might also be contributory. In conclusion, the present observations endorse the correlation between disease severity and oxygen radical production by neutrophils in asthmatic patients and suggest that oxygen metabolites may playa direct or indirect role in the modulation of airway inHammation. REFERENCES
1 Juniper ET, Firth PA, Hargreave FE. Airway responsiveness to histamine and methacholine: relationship to minimum treatment control symptoms of asthma. Thorax 1981; 36:575-79 2 Chung leF. Role of inOammation in the hyperreactivity of the airways in asthma. Thorax 1986; 41:657-62 3 Metzger WJ, Richerson HB, Worden Ie, Monick M, Hunningbake GW Bronchoalveolar lavage of allergic patients following allergen bronchoprovocation. Chest 1986; 89:477-83 4 Metzger WJ, Zavala D, Richerson HB. Local allergen cballenge and bronchoalveolar lavage of allergic asthmatic lungs. Am Rev Respir Dis 1987; 135:433-40 5 Gin ~ Kay AB. The effect of corticosteroids on monocyte and neutrophil activation in bronchial asthma. J Allergy Clio Immunoll985; 76:675-82 6 Gin ~ Shaw 8J, Kay AB. Airways reversibility after predonisolone therapy in chronic asthma is associated with alteration in leukocyte function. Am Rev Respir Dis 1985; 132:1199-1203 7 Ann J~ Walport JJ, Loe TH. Expression of complement receptors type 1 (CRl) and type 3 (CR3) on circulating granulocytes in experimentally provoked asthma. J Allergy Clin Immunol 1989; 83:649-55 8 WangSR, YangCM, Wang SSM, HanSH,ChiangBN. Enhancement of A 23187-induced production of the slow-reacting substance on peripheral leukocytes from subjects with asthma. J Allergy Clin Immunoll986; 77:465-71 9 Mita H, Yui Y, Taniguchi N, Yasuda H, Shida T. Increased activity of 5-lipooxygenase in polymorphonuclear leukocytes from asthmatic patients. Life Sci 1985; 37:W1-14 10 80rgeat ~ Samuelsson B. Arachidonic acid metabolism in polymorphonuclear leukocytes: effects of ionophone A 23187. Proc Natl Acad Sci USA 1979; 76:2148-52 11 Goldstein 1M, Malmsten CL, Samuelsson B, Weissman G. Prostaglandins, thromboxanes, and polymorphonuclear leukoCHEST I 100 I 5 I NOVEMBER, 1991
1321
cytes. Inflammation 1977; 2:309-17 12 Babior BM. Oxygen-dependent microbial killing by phagocytes. N Engl J Med 1978; 298:721-25 13 Shasby DM, Vandenthuysen KM, Tate RM, Shasby SS, McMurtry I, Repine JE. Granulocytes mediate acute edematous lung injury in rabbits and in isolated rabbit lungs perfused with PMA: role ofoxygen radicals. Am Rev Respir Dis 1982; 125:44347 14 Petrone WF, English DK, Wong K, McCord JM. Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc Natl Acad Sci USA 1980; 1159-63 15 Boyum A. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at Ig. Scand J Clin Lab Invest 1968; 21(suppl 97):77-89 16 McCord JM, Fredorich I. Superoxide dismutase. J BioI Chern 1969; 224:6049-55 17 Massey ~ The microestimation of succinate and the extinction
1322
18
19
20
21
22
coefficient of cytochrome C. Biochem Biophys Acta 1969; 34:225-56 Marone G, Thomas LL, Lichtenstein LM. The role of agonists that active adenylate cyclase in the control of cAHP metabolism and enzyme release by human polymorphonuclear leukocytes. J Immunol 1980; 125:2277-83 Neijens HJ, Raateep RE, Degenhart J, Duiverman EJ, Kerrebijn KF. Altered leukocyte response in relation to the basic abnormality in children with asthma and bronchial hyperresponsiveness. Am Rev Respir Dis 1984; 130:744-47 O'Byrne PM, Walters EH, Gold BD. Neutrophil depletion inhibits airway hyperresponsiveness induced by ozone exposure. Am Rev Respir Dis 1984; 130:214-19 Holtzman MJ, Fabbri LM, O'Byrne PM, Gold BD, Aizawa H, Walters EH, et ale Importance of airway inflammation for hyperresponsiveness induced by ozone. Am Rev Respir Dis 1983; 127:686-90 Seltzer J, Bigby BG, Stulbarg M. Ozone-induced change in bronchial reactivity to methacholine and airway inflammation in humans. J Appl Physiol 1986; 60: 1321
Free Radicals in Airway Obstruction in Asthma (KsnszsW8 et 81)