(Chest. 2000;118:180-182.)
© 2000
American College of Chest Physicians
Clara Cell Secretory Protein*
Levels in BAL Fluid After Smoking Cessation
Olof Andersson, MD, PhD;
Tobias N. Cassel, BSc;
C. Magnus Sköld, MD, PhD;
Anders Eklund, MD, PhD, FCCP;
Johan Lund, PhD and
Magnus Nord, MD, PhD
*
From the Departments of Lung Medicine (Dr. Andersson), Medical Nutrition (Mr. Cassel and Dr. Nord), and Respiratory Medicine (Drs. Sköld and Eklund), Karolinska Institute, Huddinge, Sweden; and the Department of Anatomy and Cell Biology (Dr. Lund), University of Bergen, Norway.
Correspondence to: Olof Andersson, MD, PhD, Department of Lung Medicine, Karolinska Institute, Huddinge University Hospital, Huddinge S-141–86, Sweden; e-mail: olle.andersson{at}mednut.ki.se
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Abstract
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Study objectives: The bronchiolar Clara cell is a major
target for tobacco smoke exposure. To improve our understanding of the
putative regenerative/repair mechanism(s) in the bronchiolar
epithelium, we measured the levels of the Clara cell secretory protein
(CCSP) in BAL fluid in healthy volunteers following smoking
cessation.
Design: BAL was performed before smoking
cessation, and at 1, 3, 6, 9, and 15 months following smoking
cessation, in eight healthy volunteers with a previous mean cigarette
consumption of 19 pack-years. The levels of CCSP in BAL fluid were
assessed in immunoblotting experiments using an antibody against human
CCSP.
Results: Significantly (p < 0.05) higher
levels of CCSP in BAL fluid were observed at 3, 6, and 9 months after
smoking cessation, while the levels of CCSP in BAL fluid at 15 months
after smoking cessation were the same as those before smoking
cessation.
Conclusions: Despite the long history of
smoking among patients in the present study group, signs of early
regeneration in the bronchiolar epithelium were noted, in that the
levels of CCSP in BAL fluid were elevated at the indicated time points
following smoking cessation. Furthermore, we propose that the insult to
the bronchiolar epithelium made by cigarette smoking caused the levels
of CCSP in the BAL fluid at 15 months after smoking cessation to return
to the levels noted before smoking cessation. The present study
suggests a role for CCSP as a marker for nonciliated bronchiolar cell
function.
Key Words: BAL • Clara cell secretory protein • human • smoking cessation
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Introduction
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The
bronchioles are a major target for tobacco smoke exposure, and a
significant decrease in the number of Clara cells is seen there in
individuals who smoke.1
In nonsmoking individuals, Clara
cells account for 8% of the cells lining the bronchioles (range, 2 to
14%).1
In BAL fluid from smokers, lower levels of the
Clara cell secretory protein (CCSP) have been demonstrated, initially
by Lund et al2
and later confirmed by
others.3
These lower levels of CCSP probably reflect
ultrastructural changes in the bronchioles, as the correlation between
levels of CCSP in BAL fluid and the number of Clara cells has
been demonstrated.4
In a previous study from our
laboratory, we demonstrated higher levels of CCSP in tracheal lavage
fluid harvested from infants with infant respiratory distress
syndrome,5
levels which possibly were a result of the
ongoing regeneration of the bronchiolar epithelium. In later stages of
the disease, with the significant dedifferentiation of the epithelium,
we saw lower levels of CCSP in tracheal lavage fluid, which further
supports the role of a well-differentiated bronchiolar epithelium for
the maintenance of CCSP levels in the airways. Bearing this in mind, we
hypothesized that smoking cessation would cause alterations in the
levels of CCSP in the BAL fluid.
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Materials and Methods
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BAL was performed before smoking cessation and at 1, 3, 6, 9,
and 15 months after smoking cessation, according to standard
guidelines, in eight healthy volunteers (seven women and one man; mean
age, 36.8 years; age range, 29 to 47 years) with mean cigarette
consumption of 19.6 pack-years (Table 1
). The results of routine physical examinations and chest radiographs
were normal in all participants. Lavage fluid was centrifuged at
250g for 10 min, and the resulting supernatant was stored at
-70°C until further analyzed. Human CCSP immunoreactivity in BAL
fluid was quantified in Western immunoblotting experiments.
Polyacrylamide gel electrophoresis was performed in the presence of
sodium dodecyl sulfate on a vertical slab gel unit (model SE 400;
Hoefer Scientific Instruments; San Francisco, CA) under nonreducing
conditions. The gels were 10%, and proteins from sodium dodecyl
sulfate-polyacrylamide gel electrophoresis were blotted onto
nitrocellulose filters and detected immunologically, as described
previously.6
Antibody-antigen complexes were visualized by
reaction with 125I-protein A and phosphoimager
plates. To permit a quantitative analysis of human CCSP in BAL fluid,
we included known amounts of purified recombinant human
CCSP7
in each Western blot immunoassay. For each specific
BAL fluid sample, the corresponding amount of CCSP in the BAL sample
was constructed from the linear part of the standard curve. Statistical
analysis was performed by the nonparametric Wilcoxon signed rank
test. Significance was accepted at p < 0.05. The study was approved
by the Ethics Committee at Karolinska Hospital, and informed consent
was obtained from all participants.
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Results
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As can be seen from Figure 1
, a small but insignificant rise in the levels of CCSP in BAL fluid,
compared to CCSP levels before smoking cessation, was noted 1 month
after smoking cessation. At 3 months after smoking cessation,
significantly higher levels (p < 0.05) of CCSP in BAL fluid were
observed in the study group. These elevated levels of CCSP in BAL fluid
remained at 6 months and 9 months after smoking cessation, but the
levels of CCSP in BAL fluid at 15 months after smoking cessation were
the same as those noted before smoking cessation.
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Figure 1.. Human CCSP immunoreactivity in BAL fluid from
eight healthy volunteers before and 1, 3, 6, 9, and 15 months after
smoking cessation. Each individual is graphically represented by a
symbol and a line. Statistical analysis was performed by Wilcoxon
signed rank test, as described in the "Methods" section.
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Discussion
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Perhaps as a consequence of its high content of xenobiotic
metabolizing enzymes,8
the Clara cells are susceptible to
injury from a wide variety of environmental contaminants. In
particular, the bronchiolar Clara cells are considered to be one of the
main targets in the mammalian lung for tobacco smoke
exposure.9
While the acute phase of the injury response to
noxious gases has been well characterized in animal
experiments,10
adaptive processes within the Clara cells
need to be further evaluated for the putative induction of refractory
cells. Irrespective of the methods applied, numerous previous reports
have been able to outline measurements of CCSP in BAL fluid as being,
perhaps, the most sensitive marker of cigarette smoking
exposure.3
Even more importantly, an inverse correlation
between the levels of CCSP in BAL fluid and the number of pack-years of
cigarette smoking has been demonstrated.3
To our
knowledge, however, this is the first report that describes the
temporal changes in the levels of CCSP in BAL fluid following smoking
cessation.
It has been shown that decreased CCSP levels in the BAL fluid of
smokers corresponds to a lower number of CCSP-synthesizing (Clara)
cells.4
In light of this, it seems likely that the
transient increase in CCSP levels after smoking cessation that was
observed in the present study reflects a regenerative process involving
the Clara cells. However, 15 months after smoking cessation, the CCSP
levels declined to the reduced levels noted before smoking cessation,
suggesting an irreversible insult to the bronchiolar epithelium from
tobacco smoke, and suggesting that the regenerative process is
incomplete. For comparison, it should be noted that, in our laboratory,
the normal mean (± SD) level of CCSP in the lavage fluid of
nonsmokers has been found to be 7.6 ± 2.6 ng/µL. In the present
study, the mean level of CCSP before smoking cessation was found to be
approximately half of that value, which is in line with previous
observations.2
3
The present study results indicate that the examination of airway
secretions for levels of CCSP in humans is a sensitive method of
detecting insults to the bronchiolar epithelium and of studying the
signs of repair. It would be of great value to extend the analysis to
the exposure of humans to other toxic lung xenobiotics (eg,
ozone, NO2, and diesel exhaust). Through the
results from human studies and those from animal and in
vitro cell culture models, it should be possible to obtain an
understanding on the cellular level of the role of Clara cells after
lung injury by inhaled toxicants.
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Acknowledgements
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We are grateful for the technical
assistance provided by Lena Nordlund-Möller.
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Footnotes
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Abbreviation:
CCSP = Clara cell secretory protein
This research was supported by the Swedish Medical Research Council
(grant No. 13115), the Swedish Medical Society, the Swedish Heart-Lung
Foundation, Swedish Match (grant No. 199831), the research foundations
"Tore Nilssons stiftelse för medicinsk forskning,"
"Stiftelsen Lars Hiertas minne," "Stiftelsen Sigurd och Elsa
Goljes minne," "Stiftelsen cystisk fibros forskningsfond," and
the Research Foundations of the Karolinska Institute.
Received for publication August 31, 1999.
Accepted for publication January 19, 2000.
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References
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Andersson, O, Noack, G, Robertsson, B, et al (1994) Ontogeny of a human polychlorinated biphenyl binding protein: level of expression in tracheal aspirates in bronchopulmonary dysplasia. Chest 105,17-22[Abstract/Free Full Text]
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Abstract
Introduction
