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* From the First Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients:
Twenty-four male patients with smoking-related stable pulmonary
emphysema without 
1-antitrypsin deficiency and nine
normal healthy volunteers.
Measurements: We measured
the pulmonary function, the inflammatory cells, and the levels of
eosinophil cationic protein (ECP), immunoreactive neutrophil
elastase-1-protease inhibitor (NE-1-PI)
complex, and interleukin (IL)-8 in sputum induced from patients with
pulmonary emphysema in its stable phase before and after treatment with
20 mg oral prednisolone per day for 2 weeks.
Results:
The eosinophil and neutrophil counts and the concentrations of ECP,
NE-1-PI complex, and IL-8 in the sputum were
significantly increased at baseline. The eosinophil count at baseline
was significantly correlated with the reversibility of airflow
obstruction following treatment, and the treatment also significantly
reduced the eosinophil numbers and ECP level in the sputum. In
contrast, the increased neutrophil number and the concentrations of
NE-1-PI complex and IL-8 at baseline did not correlate
with the reversibility and were not affected by treatment.
Conclusions: These findings suggest that the eosinophilic inflammation, not neutrophilic inflammation, in the airway is involved in the reversible part of the airflow obstruction in response to glucocorticoids in patients with pulmonary emphysema.
Key Words: corticosteroid • eosinophil • eosinophil cationic protein • IL-8 • induced sputum • neutrophil • neutrophil elastase • pulmonary emphysema
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, to examine the effects of glucocorticoids on
airflow inflammation and their association with the reversibility of
airway obstruction in response to glucocorticoid treatment in COPD, we
measured pulmonary function, the inflammatory cells, and the levels of
ECP, immunoreactive neutrophil elastase-1-protease
inhibitor (NE-1-PI) complex, and interleukin (IL)-8 in
sputum induced from patients before and after a short course of oral
prednisolone therapy. We studied patients with chronic pulmonary
emphysema in its stable phase because the chronic bronchitis of COPD
patients is sometimes difficult to discriminate clinically from chronic
asthma. Asthma patients often show a poor reversibility of
bronchoconstriction in response to a ß2-agonist.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin deficiency were
recruited from our outpatient clinic (Table 1
).
They had a smoking history of > 30 pack-years. Pulmonary emphysema
was diagnosed based on a clinical history of exertional dyspnea,
pulmonary function characterized by irreversible airway obstruction
(FEV1/FVC < 70%; FEV1 < 70% predicted
value after inhalation of bronchodilator), lung hyperinflation,
decreased gas transfer, and anatomic emphysema on high-resolution CT.
Patients with any history of asthma or variability in symptoms, and
patients who showed reversibility 
10% predicted FEV1
after 20 µg inhaled procaterol hydrocloride, had taken inhaled or
oral steroids, or had suffered a respiratory tract infection or
exacerbation of their airway disease in the previous 6 weeks were
excluded. All were treated with regular inhalation of a bronchodilator
and slow-releasing theophylline for > 6 months before the study. Nine
normal healthy volunteers (seven men and two women) ranging in age from
24 to 50 years, with a mean age of 33 years were recruited; all were
nonsmokers, nonatopic, and showed no abnormalities in pulmonary
function tests. The study was approved by the local research ethics
committee, and all patients gave informed consent.
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1-PI
complex, and IL-8.
Sputum Collection and Analysis
Sputum was induced by having the subject inhale hypertonic
saline solution as previously described.16
,17
Briefly,
prior to the induction of sputum, all subjects inhaled a
ß2-agonist. The hypertonic saline solution was nebulized
with an ultrasonic nebulizer (NE-V10B; Omron; Tokyo, Japan) at maximum
output for 5-min periods, up to 20 min. The concentration of saline
solution was increased at 10-min intervals, from 3.5 to 4.5%. If the
FEV1 fell by > 10% from the postbronchodilator value,
the concentration of saline solution was not increased. If the
FEV1 fell by > 20% or if troublesome symptoms occurred,
the nebulization was discontinued. The subjects were asked to rinse
their mouths and throats, and then to try to cough sputum into a
sterile plastic container. The nebulization was continued for at least
10 min and stopped after 20 min or earlier if a 2-mL sputum sample
of good quality was obtained. The volume of the induced sputum samples
was determined. A small aliquot of the sample containing an adequate
mucous plug for cell counting was overlaid with an equal volume of
Hanks' balanced salt solution containing 1 mM dithiothreitol (Sigma
Chemicals; Poole, UK) and incubated at 37°C for 15 min. The incubated
suspension was washed with Hanks' balanced salt solution two times.
After the residual mucus was removed by filtering the suspension with
gauze, the eluant was provided for total and differential cell counts.
The total cell count except for squamous cells was determined using a
standard hemocytometer, and normalized for weight and expressed as
cells x 105/g wet weight sputum. Cell smears were
prepared with a centrifuge (Autosmear; Sakura; Tokyo, Japan) and
stained with May-Grünwald-Giemsa stain for the differential cell
count, which was carried out by a "blinded" investigator. The
slides were coded and 500 cells were counted for the differential
leukocyte count. A sample was considered adequate when the percentage
of squamous cells was < 20%. The results of the differential
leukocyte counts are expressed as a percentage of nucleated cells
excluding squamous and epithelial cells. To the remaining sputum
samples was immediately added three volumes of normal saline solution,
and the samples were sonicated and centrifuged at 2,000 g
for 20 min. The supernatant was aspirated and frozen at -80°C. We
measured the concentration of ECP using an 125I-ECP
radioimmunoassay kit (Pharmacia Diagnostics; Uppsala, Sweden), albumin
by laser nephelometry, and immunoreactive NE-1-PI
complex and IL-8 using enzyme-linked immunosorbent assay kits purchased
from two companies, respectively (Merck Co; Darmstadt, Germany; and
Toray Fuji Bionics; Tokyo, Japan).
Data Analysis
The values shown in the text and tables are mean ± SEM. The
data distribution of the variables in each group was first assessed
using Bartlett's test. When the data for the variables showed a normal
distribution, the comparison was performed with a one-way analysis of
variance, and then multiple comparisons were performed by the
Tukey-Kramer method. When the data for the variables did not show a
normal distribution, the variables were compared using the
Kruskal-Wallis test, and then multiple comparisons among groups were
performed by the nonparametric Tukey-Kramer method. The correlation
between variables was examined by calculating Pearson's product
correlation coefficient. A p value of < 0.05 was considered
significant for all statistical tests.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Analysis and Chemical Substances in Induced Sputum at Baseline
The results of the cell analysis and the concentration of chemical
substances at baseline are shown in Table 2
.
The total cell counts per 1 g of sputum in the patients with
pulmonary emphysema were significantly higher compared with those from
the healthy control subjects. The sputum from the patients contained
significantly greater relative and absolute cell numbers of both
neutrophils and eosinophils compared with the sputum from the healthy
control subjects. Some patients showed an increase in the concentration
of albumin, but there was no significant difference. The concentrations
of ECP, immunoreactive NE-1-PI complex, and IL-8 in the
sputum from the patients showed significantly higher levels compared
with those from the healthy control subjects. When the concentrations
of ECP, NE-1-PI complex, and IL-8 were corrected by the
concentration of albumin (ECP/albumin, NE-1-PI
complex/albumin, IL-8/albumin), significant increases in these chemical
substances were also observed. The relative eosinophil numbers in
the sputum were positively correlated with the concentration of
ECP/albumin (r = 0.44, p < 0.05). However, no significant
correlation between each differential cell count and the concentration
of NE-1-PI complex or IL-8 was obtained.
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1-PI complex, and IL-8 in the
sputum at baseline were obtained. The prednisolone treatment
significantly reduced the increased relative and absolute eosinophil
numbers in the sputum (Table 3
),
but did not reduce the increased neutrophil numbers obtained at
baseline. The increased ECP and ECP/albumin levels in the sputum at
baseline were also significantly decreased following prednisolone
treatment. In addition, in the eight patients who showed no improvement
in FEV1, the eosinophil numbers and ECP/albumin levels in
sputum did not change following treatment (eosinophil, 1.2 ± 0.5%
at baseline vs. 0.8 ± 0.2% after treatment; ECP/albumin,
3.77 ± 1.95 x 10-3 at baseline vs
2.34 ± 0.62 x 10-3 after treatment) (Fig 3
).
However, in 11 patients who showed an improvement in FEV1,
the eosinophil numbers and ECP/albumin levels decreased following
treatment (eosinophil, 9.7 ± 3.6% at baseline vs 1.0 ± 0.3%
after treatment [p < 0.05]; ECP/albumin,
7.40 ± 3.24 x 10-3 at baseline vs
1.85 ± 0.44 x 10-3 after treatment [p = 0.09])
(Fig 3
). The increased NE-1-PI complex and IL-8 levels
seen at baseline did not show significant decreases following
treatment.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eosinophils have been reported in the airways of patients with COPD.8 ,9 ,10 The concentrations of ECP and eosinophil peroxidase, which are derived from activated eosinophils, are also highly correlated20 with the eosinophil numbers in the airways of COPD patients.12 ,21 We also observed significant increases in eosinophil numbers and ECP levels in induced sputum in patients with stable pulmonary emphysema. We recently reported significant increases in eosinophil numbers (18.2 ± 3.6%) and ECP levels (526.1 ± 128.2 µg/L) in sputum induced from patients with asthma.22 The eosinophil counts in sputum, even when corrected by the total cell per gram of sputum, are not so high in patients with emphysema compared with those of asthma patients; however, the concentrations of ECP in the sputum of COPD patients showed markedly higher levels. Keatings and Barnes21 obtained similar data in a comparison of asthma and COPD patients. The ECP levels corrected by the concentration of albumin in the sputum from COPD patients were significantly correlated with eosinophil numbers at baseline, and the treatment with oral prednisolone significantly reduced the ECP levels together with the decrease in eosinophil numbers. These findings suggest that the increased ECP is degranulated from eosinophils, and that the eosinophils in the airway of pulmonary emphysema patients are highly activated.
A short course of oral glucocorticoids administered to patients with COPD in its stable phase has been reported to produce an improvement in airway obstruction in some but not all patients.9 ,10 ,11 When compared with the patients who show no significant responses to glucocorticoids, responders have significantly larger numbers of eosinophils and higher levels of ECP in their BALF.12 In the present study, 12 patients (50%) showed an increase in FEV1 of > 12% from baseline values, and 5 patients (21%) showed an increase in FEV1 of > 200 mL following the administration of 20 mg oral prednisolone for 2 weeks. The reversibility of airway obstruction following the treatment was significantly positively correlated with the eosinophil numbers in the sputum induced at baseline. The treatment significantly reduced eosinophil numbers and the concentrations of ECP and ECP/albumin in the sputum. In addition, in the patients who showed an improvement in FEV1 following the treatment, the eosinophil numbers were significantly decreased and the ECP/albumin level had a tendency to be reduced, whereas in the patients who showed no improvement in FEV1, the eosinophil numbers and ECP/albumin levels were lower at baseline and did not change following the treatment. These findings suggest that the activated eosinophils are involved in the airway inflammation and contribute to the airflow obstruction, which shows an improvement in response to glucocorticoids in patients with pulmonary emphysema.
In COPD, the consequent protease burden, in addition to reduced antiprotease capacity, is contributory to the development of irreversible airflow obstruction.1 The enzyme most implicated has been neutrophil elastase. Yoshioka et al23 found that neutrophil elastase in BALF is significantly increased in smokers who are potentially developing emphysema, but not in smokers who are not. IL-8, the main actions of which are neutrophil recruitment and activation,24 may play a part in neutrophil recruitment in COPD.25 We found here that the patients with pulmonary emphysema showed higher neutrophil elastase and IL-8 levels in induced sputum, and these chemical substances may be implicated in the airway inflammation mediated by neutrophils in the pathogenesis of emphysema. A significant reduction in the chemotactic activity of neutrophils and an increase in neutrophil elastase inhibitory capacity, including secretory leukoprotease inhibitor in the sputum of COPD patients following the treatment of glucocorticoids, have been reported,13 ,14 and glucocorticoids were found to inhibit IL-8 transcription in human airway epithelial cells in vitro.26 In the present study, however, the short course of oral prednisolone administration did not reduce the neutrophil numbers or the concentrations of neutrophil elastase and IL-8 in the sputum, as has been reported previously.13 ,15 In addition, the neutrophils and these substances did not show any correlation with the reversibility of airflow obstruction in response to prednisolone treatment. These findings suggest that the inflammation concerned with neutrophils may be important in the development of the irreversible pathogenesis of emphysema, but not involved in at least the reversible part of airflow obstruction in response to a short course of systemic glucocorticoid administration.
In summary, the eosinophil and neutrophil numbers and the
concentrations of ECP, NE-1-PI complex, and IL-8 in
induced sputum were significantly increased in patients with stable
pulmonary emphysema. The eosinophil numbers at baseline were
significantly correlated with the increases in FEV1
following oral prednisolone treatment for 2 weeks, and the treatment
also significantly reduced the eosinophil numbers and ECP level in the
sputum. However, the increased neutrophil numbers and the
concentrations of NE-1-PI complex and IL-8 at baseline
did not correlate with the increases in FEV1 following the
treatment, and did not change after the treatment. These findings
suggest that eosinophilic inflammation, not neutrophilic inflammation,
in the airway is chiefly involved in the reversible part of the airflow
obstruction in response to glucocorticoids in some patients with
pulmonary emphysema, and that sputum eosinophilia could be an indicator
for predicting the improvement in airflow obstruction following
glucocorticoid treatment.
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Footnotes |
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Correspondence to: Keishi Kubo, MD, First Department of Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan; e-mail: keishik@hsp.md.shinshu-u.ac.jp
Abbreviations: BALF = BAL fluid;
ECP = eosinophil cationic protein;
NE-1-PI = neutrophil
elastase-1-protease inhibitor
Received for publication June 10, 1998. Accepted for publication October 27, 1998.
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References |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 153,530-534[Abstract]
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