Serum surfactant protein D is steroid

sensitive and associated with

exacerbations of COPD
D.A. Lomas*, E.K. Silverman
#
, L.D. Edwards
"
, N.W. Locantore
"
, B.E. Miller
+
,
D.H. Horstman
"
and R. Tal-Singer
+
on behalf of the Evaluation of COPD
Longitudinally to Identify Predictive Surrogate Endpoints study investigators
ABSTRACT: Surfactant protein (SP)-D is a lung-derived protein that has been proposed as a
biomarker for inflammatory lung disease.
Serum SP-D was evaluated as a biomarker for components of chronic obstructive pulmonary
disease (COPD) in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate
Endpoints (ECLIPSE) cohort and its response assessed to the administration of the anti-
inflammatory agent prednisolone.
The median level of serum SP-D was significantly elevated in 1,888 individuals with COPD
compared to 296 current and former smokers without airflow obstruction (121.1 and
114.3 ng?mL
-1
, respectively; p50.021) and 201 nonsmokers (82.2 ng?ml
-1
; p,0.001). There was
no correlation with the severity of COPD. Individuals with COPD who had a serum SP-D
concentration that was greater than the 95th percentile of nonsmokers (175.4 ng?mL
-1
) showed an
increased risk of exacerbations over the following 12 months (adjusted OR 1.30; 95% CI 1.03
1.63). Treatment with 20 mg?day
-1
prednisolone for 4 weeks resulted in a fall in serum SP-D levels
(126.0 to 82.1 ng?mL
-1
; p,0.001) but no significant change in post-bronchodilator forced
expiratory volume in 1 s.
Serum SP-D concentration is raised in smokers and may be useful in identifying individuals who
are at increased risk of exacerbations of COPD. It may represent an intermediate measure for the
development of novel anti-inflammatory agents.
KEYWORDS: Biomarker, bronchitis, emphysema, exacerbation, inflammation, prednisolone
C
hronic obstructive pulmonary disease
(COPD) is a multicomponent condition
that is characterised by airways obstruc-
tion, emphysema, mucus hypersecretion and
systemic disease that vary in proportion between
affected individuals [1, 2]. The development of
disease is intimately associated with the inhala-
tion of noxious agents and, in particular, cigarette
smoke [3]. There is clearly an urgent need for a
simple biomarker that can be used in the
diagnosis of COPD and to assess prognosis and
the effectiveness of therapeutic interventions.
Biomarkers have been assessed in urine, blood,
sputum, bronchoalveolar lavage fluid, skin and
exhaled breath condensate, but none is widely
accepted to be reproducible and to discriminate
between smokers with and without airflow
obstruction [4]. Moreover, none has proved
useful as a robust end-point in clinical trials.
Surfactant protein (SP)-D is a large hydrophilic
protein that is a member of the collagen-containing
C-type lectins or collectins [5]. Its structure is based
on a triple-helical collagen region and a C-terminal
homotrimeric lectin or carbohydrate recognition
domain. Four of the homotrimeric subunits of SP-D
are assembled via their N-terminal region into a
520-kDa dodecameric structure that can further
oligomerise to formmultimers. SP-Dis found in the
endoplasmic reticulumof type II pneumocytes and
the secretory granules of Clara cells [6]. It makes an
important contribution to surfactant homeostasis
and pulmonary immunity [5]. SP-D plays a role in:
protecting against viral infection; clearance of
bacteria, fungi and apoptotic cells; and resolution
of inflammation [7]. Mice that lack SP-D develop
chronic inflammation and emphysema that can be
prevented by administration of truncated recom-
binant human SP-D [8]. Since SP-D is synthesised
AFFILIATIONS
*Dept of Medicine, University of
Cambridge, Cambridge Institute for
Medical Research, Cambridge, UK.
#
The Channing Laboratory and
Pulmonary and Critical Care Division,
Brigham and Womens Hospital and
Harvard Medical School, Boston, MA,
"
GlaxoSmithKline, Research Triangle
Park, NC, and
+
GlaxoSmithKline, King of Prussia,
PA, USA.
CORRESPONDENCE
D.A. Lomas
Dept of Medicine, University of
Cambridge
Cambridge Institute for Medical
Research
Wellcome Trust/MRC Building
Hills Road
Cambridge
CB2 0XY
UK
E-mail: dal16@cam.ac.uk
Received:
Oct 15 2008
Accepted after revision:
Jan 02 2009
First published online:
Jan 22 2009
European Respiratory Journal
Print ISSN 0903-1936
Online ISSN 1399-3003
EUROPEAN RESPIRATORY JOURNAL VOLUME 34 NUMBER 1 95
Eur Respir J 2009; 34: 95102
DOI: 10.1183/09031936.00156508
CopyrightERS Journals Ltd 2009
c
predominantly within the respiratory tract, it has been evaluated
as a potential biomarker in small numbers of individuals with
community-acquired pneumonia [9], drug-induced lung disease
[10, 11], interstitial fibrosis [12] and allergic bronchopulmonary
aspergillosis in cystic fibrosis [13]. Levels are reduced in
bronchoalveolar lavage fluid from individuals with COPD [14],
and there was a weak inverse relationship between serum SP-D
level and forced expiratory volume in 1 s (FEV1) in 23
individuals with advanced COPD [15]. The utility of serum
SP-D as a biomarker for components of the COPD phenotype
was evaluated and the effect assessed of oral corticosteroids on
levels of this biomarker.
MATERIALS AND METHODS
The population under study was the Evaluation of COPD
Longitudinally to Identify Predictive Surrogate Endpoints
(ECLIPSE; study identification number SCO104960;
Clinicaltrials.gov identifier NCT00292552) cohort.
The aims and operational aspects of the ECLIPSE cohort have
been described elsewhere [16, 17]. Briefly, the ECLIPSE study
is a 3-yr multicentre longitudinal observational study to
identify novel end-points in COPD. Individuals aged
4075 yrs were recruited to the study if they had a smoking
history of o10 pack-yrs, a post-bronchodilator FEV1/forced
vital capacity (FVC) ratio of f0.7 and Global Initiative for
Chronic Obstructive Lung Disease (GOLD) stage II (FEV1
5080% of the predicted value), III (FEV1 3050% pred) or IV
(FEV1 ,30% pred) COPD [3]. Smoking (o10 pack-yrs) and
nonsmoking (,1 pack-yr) control subjects were enrolled if
they were aged 4075 yrs and exhibited normal lung function
(post-bronchodilator FEV1 of .85% pred and FEV1/FVC of
.0.7). Individuals recruited to the study were genotyped for
a
1
-antitrypsin deficiency. Six PiZZ and 11 PiSZ individuals
were identified and excluded from the analysis.
All subjects underwent standardised spirometry following
180 mg (2 puffs) salbutamol [18], with reversible airflow
obstruction being defined as an increase in FEV1 of 15% and
o200 mL. All subjects were offered a low-dose computed
tomography (CT) scan of the chest in order to exclude non-
COPD-related disease and evaluate the severity and distribu-
tion of emphysema. The CT scans were evaluated at the central
imaging unit of the University of British Columbia (Vancouver,
BC, Canada). The extent of emphysema was assessed in two
ways. First, it was independently scored by two radiologists
who were blind to the individuals lung function. Emphysema
was reported as trivial, mild, moderate, severe and very severe
if it affected ,5, 525, 2550, 5075 and .75% of the lungs,
respectively. A consensus reading was obtained when there
was a difference of more than one emphysema category
between the two observers. Otherwise, the mean of the two
readings was used in the analysis. Second, emphysema was
assessed by the percentage of the lung with attenuation of
,-950 HU using Pulmonary Workstation 2.0 software (VIDA
Diagnostics, Inc., Iowa City, IA, USA).
Assessment of exacerbations in the ECLIPSE cohort
COPD subjects were asked about exacerbations 3, 6 and
12 months after enrolment in the study. In addition, they were
contacted by telephone every month by the study staff and
asked about details of exacerbations during the previous
month. Specifically, subjects were asked whether they had
been unwell in the last month, whether they had seen a doctor
or been to hospital and whether they had taken any medication
for exacerbations (oral corticosteroids or antibiotics). The data
were analysed 12 months after enrolment into the study.
Effect of oral corticosteroids on serum surfactant protein D
in individuals with chronic obstructive pulmonary disease
The effects of oral prednisolone were investigated in a separate
study (Clinicaltrials.gov identifier NCT00379730).
Current/former smokers aged 4080 yrs with a post-salbuta-
mol FEV1 of 3080% pred and chronic bronchitis (n589) were
recruited to a study that was separate from the ECLIPSE study.
Chronic bronchitis was defined as daily cough productive of
sputum for 3 months for 2 yrs successively [19]. Individuals
were excluded if they had had an exacerbation of COPD
requiring steroid or antibiotics in the month prior to the 28-day
screening period or were taking oral or inhaled steroids for
.14 days consecutively in the 6 months prior to screening. All
subjects were offered a CT scan of the chest, which was
performed and analysed as detailed for the ECLIPSE study.
Individuals were randomised to receive either placebo or
20 mg?day
-1
prednisolone for 4 weeks, 10 mg?day
-1
predniso-
lone for 1 week and 5 mg?day
-1
prednisolone for 1 week. Both
groups were followed-up for 2 weeks following cessation of
treatment. Serum samples were taken at baseline and every
2 weeks throughout the study.
Measurement of serum surfactant protein D
Whole blood was collected into vacutainer tubes at the start of
the studies. Serum was prepared by centrifugation for
1015 min at 1,5006g. The serum was collected and stored at
-80uC until analysed. Serum SP-D was measured by operators
who were blind to an individuals lung disease or treatment
group using a colorimetric sandwich immunoassay method
(BioVendor, Heidelberg, Germany) according to the manufac-
turers instructions. Samples were routinely tested at 5-fold
dilution with the dilution buffer supplied by the manufacturer.
Samples with out-of-range results were retested at higher
dilution. The concentration of SP-D in the diluted samples was
interpolated from the standard curve of recombinant human
SP-D (molecular mass 41 kDa) and then corrected for the
dilution factor. The assay had a validated range of 1.56
100 ng?mL
-1
, with an intra-assay coefficient of variation and
relative error of 1.984.06% and -7.32 -1.40%, respectively,
and an inter-assay coefficient of variation and relative error of
4.805.84% and -12.22 -2.46%, respectively.
Statistical analysis
The reproducibility of SP-D measurement in the ECLIPSE
cohort was assessed through BlandAltman plots [20]. Owing
to the non-normality of SP-D values identified by Shapiro
Wilk and KolmogorovSmirnov tests, all SP-D measurements
in the ECLIPSE cohort were logarithmically transformed prior
to analysis. All comparisons between subject groups were then
conducted by ANOVA based on the transformed values.
Spearmans correlation coefficients (based on ranks) were
calculated for correlations between SP-D level and clinical
parameters. In the prednisolone study, the effect of predniso-
lone on serum SP-D levels and FEV1 was analysed by
COPD AND SMOKING-RELATED DISORDERS D.A. LOMAS ET AL.
96 VOLUME 34 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL
ANCOVA, adjusting for baseline value and study site. In both
studies, ANOVA and CochranMantelHaenszel tests were
used to compare subject groups. SAS1 Version 8.2 (SAS
Institute, Inc., Cary, NC, USA) was used to carry out all
analyses.
Ethics
The studies were conducted in accordance with the
Declaration of Helsinki and Good Clinical Practice guidelines,
and were approved by the relevant ethics and institutional
review boards at the participating centres.
RESULTS
Assessment of surfactant protein D in the ECLIPSE cohort
Serum SP-D was measured in 1,888 individuals with COPD
from the ECLIPSE cohort, 296 smoking controls with no
airflow obstruction and 201 nonsmoking controls (table 1;
fig. 1a). Median levels of serum SP-D were higher in the
mixture of current and former smokers with COPD compared
to those without COPD (121.1 and 114.3 ng?mL
-1
, respectively;
p50.021), and in smokers compared to nonsmokers with no
airflow obstruction (114.3 and 82.2 ng?mL
-1
, respectively;
p,0.001). The serum SP-D concentration was similar in males
and females who were nonsmokers (80.8 and 83.3 ng?mL
-1
;
n574 and 127, respectively), smoker controls (118.3 and
109.5 ng?mL
-1
; n5161 and 135, respectively; p50.732) and
those with COPD (123.3 and 117.0 ng?mL
-1
; n51,222 and 666,
respectively; p50.081). Serum SP-D levels were not associated
with COPD disease severity as defined by GOLD status. There
was no difference in serum SP-D level in individuals with
COPD or in smoker controls who reported chronic bronchitis
compared with those who did not have this symptom.
Moreover, there was no correlation between serum SP-D level
and either the radiologists emphysema score or areas of low
attenuation on the CT scan (,-950 HU). There were weak
correlations between serum SP-D level and age (r50.11;
p,0.001) and body mass index (BMI) (r5-0.13; p,0.001).
Twin studies have shown that serum levels of SP-D are
elevated by smoking [21]. Levels of SP-D were, therefore,
analysed in groups divided into current and former smokers
(fig. 1b). Serum levels of SP-D were higher in current than in
former smoker controls, but were significantly higher in both
current and former smokers diagnosed with COPD (p50.024
and 0.001, respectively). The effect of smoking was apparent
across all severities of COPD, as defined by GOLD status.
Serum surfactant protein D and risk of exacerbations of
chronic obstructive pulmonary disease
Data were available for 2,189 (92%) of the 2,385 individuals
after 12 months of follow-up. There were 2,351 exacerbations
(1,446 and 905 in former and current smokers, respectively), as
defined by episodes of worsening symptoms that were self-
managed by the subject. These were reported by 1,093
individuals with COPD (670 and 423 former and current
smokers, respectively; the range of exacerbations in any
individual was 111). There was no effect of current smoking
on the incidence of exacerbations (56.9 and 58.7% in current
and former smokers, respectively) and a weak negative
correlation between the incidence of exacerbations and
percentage predicted FEV1 (r5-0.15; p,0.001). Baseline serum
SP-D level was not associated with either decline in FEV1
(r50.004; p50.866), percentage predicted FEV1 (r50.004;
p50.837) or FVC (r5-0.033; p50.117). The median serum
SP-D concentration was similar between those individuals who
had had one or more exacerbations during 12 months of
follow-up and those who had had no exacerbations (121.5
versus 120.3 ng?mL
-1
; n51,093 and 795, respectively; p50.062).
Moreover, there was no correlation between serum SP-D level
and the number of exacerbations reported during the 12-month
follow-up or whether an individual required hospitalisation
for an exacerbation. Although only a small number of
individuals died during the first 12 months of the ECLIPSE
study, there was a trend towards higher baseline median SP-D
concentrations in those individuals who died during follow-up
TABLE 1 Assessment of serum surfactant protein (SP)-D in individuals with and without chronic obstructive pulmonary disease
(COPD)
COPD subjects Smoker controls Nonsmoker controls p-value
#
Subjects n 1888 296 201
Age yrs 63.47.2 54.78.9 53.28.6 ,0.001
Males n (%) 1222 (65) 161 (54) 74 (37) 0.001
Smoking history pack-yrs 49.227.3 32.022.1 0.40.5 ,0.001
Current smoker n (%) 746 (40) 201 (68) 0 (0) ,0.001
FEV1 L 1.40.5 3.40.8 3.30.8 ,0.001
FEV1 % pred 48.715.5 108.612.1 114.814.0 ,0.001
FEV1/FVC 0.450.11 0.790.05 0.810.05 ,0.001
CT scans n 1496 260 165
Low attenuation area
"
% 16.911.8 2.22.9 3.94.0 ,0.001
SP-D ng?mL
-1
121.1 (84.9174.2) 114.3 (75.6162.3) 82.2 (56.1117.7) 0.021
Data are presented as meanSD or median (interquartile range) unless otherwise indicated. The lung function measurements followed the administration of 180 mg
salbutamol. The number of computed tomography (CT) scans is the number of scans available for qualitative analysis to assess the percentage of the lungs with a density
of ,-950 HU. FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; % pred: percentage of the predicted value.
#
: COPD subjects versus smoker controls
(p,0.001 for COPD subjects versus nonsmoker controls for all parameters);
"
: ,-950 HU.
D.A. LOMAS ET AL. COPD AND SMOKING-RELATED DISORDERS
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 34 NUMBER 1 97
compared to those who remained alive (138.8 and
120.9 ng?mL
-1
; n540 and 1,848, respectively; p50.125).
Serum SP-D concentration was assessed as a continuous
variable in a multivariate model for its ability to predict the
occurrence of at least one exacerbation during the 12-month
follow-up. The results showed an OR of 1.22 (95% CI 1.071.39)
for exacerbations for each 100 ng?mL
-1
increase in SP-D level
after adjusting for sex, percentage predicted FEV1, reversibility
and those individuals taking inhaled corticosteroids. This was
more marked if the analysis was restricted to those individuals
with a baseline SP-D level in the upper quartile (OR 1.42; 95%
CI 1.021.97). The effect may result from a small number of
very high outliers. However, the findings were even more
marked if the outliers with SP-D concentrations above the 99th
percentile (382.7 ng?mL
-1
) were excluded (OR 1.58; 95% CI
1.022.44). These results may simply reflect those individuals
who reported exacerbations prior to enrolment in the study.
The analysis was, therefore, repeated in the subset of
individuals who did not report any exacerbation during the
year prior to enrolment in the study. Serum SP-D concentration
remained associated with an increased risk of exacerbations
(OR 1.23; 95% CI 1.021.49). The results were unchanged if the
analysis was repeated with either diuretic or b-blocker
medication being included as confounding factors.
The 95th percentile of serum SP-D level in the nonsmokers was
175.5 ng?mL
-1
. This concentration was used to categorise
subjects with COPD as having either high or low levels of
serum SP-D. The OR for exacerbations of COPD was 1.30 (95%
CI 1.031.63) in individuals with high serum levels of SP-D
after adjusting for sex, percentage predicted FEV1 and those
individuals taking inhaled corticosteroids (neither age, smok-
ing status, smoking history in pack-years nor reversibility were
significant predictors in this model). Repeating this analysis
with the 75th percentile of serum SP-D concentration for COPD
subjects (174.2 ng?mL
-1
) as the cut-off for categorising the
subjects with COPD gave similar results (OR 1.28; 95% CI 1.02
1.61). Similar results were also obtained if exacerbations were
defined based on the requirement for antibiotics (,80% of all
exacerbations; OR 1.31; 95% CI 1.051.64).
Assessment of the reproducibility of serum surfactant
protein D
It is important to know whether serum SP-D level is a
reproducible biomarker, and so it was measured in an age-
matched subgroup of 195 individuals with COPD, 36 smoker
controls and 36 nonsmoking controls selected from the
ECLIPSE cohort (table 2). The individuals with COPD and
smoker controls were all former smokers in order to reduce the
variability associated with smoking status. SP-D levels were
reproducible in nonsmokers, former smokers without airflow
obstruction and across all severities of COPD when measured
over a period of 3 months (coefficient of repeatability
70.20 ng?mL
-1
; variability 26%) (fig. 2; table 2).
Effect of prednisolone on serum surfactant protein D in
individuals with chronic obstructive pulmonary disease
Serum levels of SP-D were similar in the ECLIPSE study
regardless of background therapy, including inhaled corticos-
teroids and long-acting b
2
-agonists. However, a modest fall in
serum SP-D concentration has been reported in individuals
with COPD following treatment with inhaled corticosteroids,
suggesting that SP-D may be a biomarker for anti-inflamma-
tory therapy [22]. The effect of administration of a systemic
corticosteroid on serum SP-D level was tested in a study
separate from the ECLIPSE study. A total of 89 current or
former smokers diagnosed with chronic bronchitis and COPD
were recruited and randomised to receive either oral pre-
dnisolone or placebo. The groups were well matched for age,
sex, lung function, degree of reversibility and smoking history
in pack-years (table 3). There were five withdrawals in the
prednisolone group and four in the placebo group during the
course of the study. Treatment with prednisolone resulted in a
1000
0
S
P
-
D

n
g

m
L
-
1
a) b)
800
600
400
200
***
***
#
NS S COPD II III IV
Controls GOLD stage
NS S COPD II III
Controls GOLD stage
Subjects Subjects
IV
l
l
l
l
l
l
l l
l
l
l
l
l
l
l
l l l l l
l
l

+
FIGURE 1. a) Serum surfactant protein (SP)-D levels in individuals with chronic obstructive pulmonary disease (COPD) and controls; and b) effect of current smoking on
SP-D levels in smoking controls and individuals with COPD (shown as a whole and divided into groups based on Global Initiative for Chronic Obstructive Lung Disease
(GOLD) classification). Vertical bars represent median and interquartile range ($: mean; #: outliers). The number of individuals (h)/current smokers (u) in each group was:
nonsmoker (NS) controls 201/0, smoker (S) controls 296/201, all COPD subjects 1,888/746, GOLD stage II COPD 846/334, GOLD stage III COPD 811/324, and GOLD stage
IV COPD 229/76 (&: former smokers). ***: p,0.001;
#
: p50.021;
"
: p50.024;
+
: p50.001.
COPD AND SMOKING-RELATED DISORDERS D.A. LOMAS ET AL.
98 VOLUME 34 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL
small increase relative to placebo in pre- and post-bronchodi-
lator FEV1 of 97 and 107 mL, respectively (fig. 3a); neither of
these changes were significant (p50.07 and 0.06, respectively).
However, treatment with prednisolone resulted in a striking
fall in serum SP-D level from 126.0 to 82.1 ng?mL
-1
at 4 weeks
(p,0.001) (fig. 3b). The concentration remained low whilst the
subjects were taking 20 mg?day
-1
prednisolone and rose as the
dose of steroids was reduced before returning to baseline
2 weeks after cessation of therapy. There was no change
(136.0 ng?mL
-1
at baseline, 135.8 ng?mL
-1
at week 4) in serum
SP-D level in those individuals who received placebo. The
effect of prednisolone was specific for SP-D as there was no
significant reduction in serum levels of other inflammatory
markers that have been reported to be elevated in COPD
(fibrinogen; interleukin-1b, -8 or -6; myeloperoxidase or matrix
metalloproteinase-9 (data not shown)).
DISCUSSION
The ECLIPSE cohort was used to evaluate serum SP-D as a
biomarker for COPD. The median serum SP-D level was
significantly higher in current and former smokers with COPD
than in those without airflow obstruction. The serum SP-D
level was similar in males and females, was unaffected by the
presence of chronic bronchitis and did not correlate with either
the radiologists emphysema score or areas of low attenuation
on the CT scan (,-950 HU). Moreover, there was no significant
increase in serum levels with increasingly severe disease (as
assessed by GOLD score). The difference in serum SP-D levels
between individuals with COPD and smoker and nonsmoker
controls, although significant, is not sufficiently large to use in
a screening test to diagnose COPD.
The largest difference in serum SP-D levels occurred between
nonsmokers and current/former smokers. Therefore, serum
SP-D is a powerful biomarker for smoking. Intrapulmonary
SP-D levels rise following the acute exposure of mice to
cigarette smoke [23], but are lower in lung lavage fluid from
individuals with COPD [14] and cystic fibrosis [24].
Approximately 75% of SP-D is found in bronchoalveolar
lavage fluid [5], and it is likely that this hydrophilic protein,
or its degradation products, leaks from the lung as a
consequence of increased vascular permeability associated
with inflammation. It is then detected within the circulation.
Thus serum SP-D reflects intrapulmonary inflammation, which
would explain the higher levels in smokers with and
without COPD.
A normal range for serum SP-D level can be derived from
nonsmoking controls. It was striking that those individuals
with COPD who had SP-D levels that were greater than the
TABLE 2 Assessment of the reproducibility of serum surfactant protein (SP)-D measurement
COPD subjects Smoker controls Nonsmoker controls p-value
#
Subjects n 195 36 36
Age yrs 64.56.0 60.87.7 59.78.8 0.002
Males n (%) 141 (72) 24 (67) 14 (39) 0.492
Smoking history pack-yrs 45.827.2 29.816.5 1.00.0 0.001
FEV1 L 1.20.5 3.20.6 3.10.7 ,0.001
FEV1 % pred 43.916.9 108.911.8 115.812.0 ,0.001
FEV1/FVC 0.400.12 0.800.06 0.800.05 ,0.001
CT scans n 178 29 32
Low attenuation area
"
% 22.613.5 4.54.4 5.45.5 ,0.001
Baseline SP-D ng?mL
-1
103.6 (77.6143.9) 96.5 (68.6121.4) 71.8 (49.199.8) 0.116
SP-D results at 3 months n 181 35 34
SP-D at 3 months ng?mL
-1
105.1 (70.5145.2) 95.5 (59.4120.0) 77.5 (49.596.6) 0.232
Data are presented as meanSD or median (interquartile range) unless otherwise indicated. The lung function measurements followed the administration of 180 mg
salbutamol. All of the smoking controls and individuals with chronic obstructive pulmonary disease (COPD) were former smokers. The number of computed tomography
(CT) scans is the number of scans available for qualitative analysis to assess the percentage of the lungs with a density of ,-950 HU. FEV1: forced expiratory volume in
1 s; FVC: forced vital capacity; % pred: percentage of the predicted value.
#
: COPD subjects versus smoker controls (p,0.001 for COPD subjects versus nonsmoker
controls for all parameters);
"
: ,-950 HU.
200
-250
D
S
P
-
D

n
g

m
L
-
1
150
100
50
0
-50
-100
-150
-200
0
Mean SP-D ngmL
-1
u
u
u
u
u
u
u
u
u
u
u u
u
u u
u
u u
u
u
u
u
u
u
u
uu
u
u u u
u
u
u
u
u
u
u
u
u
u
u
uu
u
u
u
u
u
u u
u
u
u u
u
u
u
u
u
uu
u u
u
u
uuu
u
u
u
u
u
u
u
u
u
u
u
u u
u
u
u
u
u
uuuu
u
u
u u u
u
u
u
u
u
uu u u u
u
u
u
u u
u
u u
u
u
u
u
u
u u
u
uuuu
u u u
uuu
u
u
u
uuu
u u
u
u
100 200 300 400 500 600 700 800 900 1000
FIGURE 2. BlandAltman plot for assessment of the reproducibility of serum
levels of surfactant protein (SP)-D at baseline and 3 months in 267 individuals with
and without chronic obstructive pulmonary disease (--------: no difference (D);
.....
: 95% limits of agreement). The bias is -2.23 and coefficient of repeatability
70.20 ng?mL
-1
.
D.A. LOMAS ET AL. COPD AND SMOKING-RELATED DISORDERS
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 34 NUMBER 1 99
95th percentile of normal controls showed a greater risk of self-
reported exacerbations. These symptoms were reported pro-
spectively and were, therefore, not dependent on recall bias.
The findings were unchanged if exacerbations were defined as
requiring the administration of antibiotics. Moreover, the risk
of exacerbations increased with increasing baseline serum
SP-D concentrations, with an even greater risk if the analysis
were confined to those individuals in the upper quartile of
baseline SP-D level. It is possible that this effect was driven by
a few outliers and thus the analysis was repeated following the
exclusion of individuals with the highest serum SP-D levels.
Again, serum SP-D was associated with exacerbations of
COPD. Finally, it is possible that serum SP-D level is affected
by comorbid conditions and so the analysis was repeated with
either diuretic or b-blocker medication being included as
confounding factors. This did not affect the results.
Previous work has shown that plasma C-reactive protein [25]
and serum amyloid A [26] are nonspecific markers of
exacerbations of COPD, with raised levels of serum amyloid
A being associated with more severe episodes [26]. However,
serum SP-D is the first biomarker that has been shown to
predict an increased risk of exacerbations of COPD in a large
prospective cohort. It is perhaps not surprising that indivi-
duals with the greatest levels of intrapulmonary inflammation
(as evidenced by raised serum SP-D levels) are at the greatest
risk of exacerbations since previous studies have shown that
the severity of exacerbations of COPD tracks with airway
inflammation [27]. Exacerbations of COPD are associated with
significant deterioration in health status [28], and thus serum
SP-D may be useful in identifying those at greatest risk
and who may, therefore, benefit from treatment with either
anti-inflammatory agents or prophylactic antibiotics. If a
biomarker is to be used to identify individuals at risk of
exacerbations then it must be stable over time. Serum SP-D
level was assessed in a different group of individuals in the
ECLIPSE cohort. The levels showed 26% variability in
nonsmokers and former smokers with and without COPD
over a 3-month interval. This variability may be higher in
individuals with higher baseline serum SP-D levels.
A biomarker that reflects the intrapulmonary inflammation of
COPD should respond to the administration of a potent anti-
inflammatory agent such as prednisolone. Indeed, there was a
rapid and marked fall in serum SP-D levels whilst individuals
150
50
L
S
M

s
e
r
u
m

S
P
-
D

n
g

m
L
-
1
75
100
0 2 4
(end
20 mg)
Time-point week
b)
0.4
-0.2
L
S
M

D
F
E
V
1

L
-0.1
0
0.1
0.2
0.3
0 2 4 6
Time-point week
l
l
l
l
n
n
n
n
a)
l
l
l
l
l
**
**
125
6
(end
run-out)
8
(end
washout)
45/44 42/43 40/39 37/39 38/38
FIGURE 3. Effect of oral corticosteroids in individuals with chronic obstructive
pulmonary disease (COPD) on: a) forced expiratory volume in 1 s (FEV1); and b)
serum surfactant protein (SP)-D level. Individuals with COPD were randomised to
prednisolone ($; &) or placebo (#; h). The prednisolone group received
20 mg?day
-1
prednisolone for 4 weeks, 10 mg?day
-1
prednisolone for 1 week and
5 mg?day
-1
prednisolone for 1 week. The effect of prednisolone on FEV1 is shown
before ($; #) and after (&; h) administration of 180 mg salbutamol. Least square
means (LSMs) were adjusted for baseline and study site in the analysis model. The
number of individuals at each time-point is shown for prednisolone/placebo in b).
Data were analysed by intention to treat and are presented as meanSEM. D:
change (from baseline (week 0)). **: p,0.001.
TABLE 3 Demographics and baseline characteristics of
individuals with chronic obstructive pulmonary
disease randomised to receive either oral
corticosteroids or placebo
Placebo Prednisolone
Subjects n 44 45
Age yrs 62.88.4 62.69.1
Males 32 (73) 35 (78)
FEV1 L 1.350.56 1.330.56
FEV1 % pred 49.615.6 46.615.1
FEV1/FVC 0.490.12 0.450.12
FEV1 reversibility % 15.114.7 18.217.9
Smoking history pack-yrs 53.435.1 48.424.5
Current smokers 21 (48) 19 (42)
Salbutamol 35 (80) 36 (80)
Ipratropium bromide 17 (39) 25 (56)
CT scans n 36 40
Emphysema on CT scan 34 (94) 36 (92)
Data are presented as meanSD or n (%) unless otherwise indicated.
Reversibility was defined as an increase in forced expiratory volume in 1 s
(FEV1) following 180 mg salbutamol. The number of individuals with any
emphysema on their computed tomography (CT) scan was determined from
the radiologists score. There were five withdrawals in the prednisolone group
and four in the placebo group. FVC: forced vital capacity; % pred: percentage
of the predicted value.
COPD AND SMOKING-RELATED DISORDERS D.A. LOMAS ET AL.
100 VOLUME 34 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL
with COPD received oral corticosteroids. Serum SP-D levels
returned to baseline following the cessation of treatment. The
change in serum SP-D levels occurred in the context of
insufficient power to detect a significant change in the
standard measures of lung function, FEV1 and FVC.
Prednisolone did not mediate its effects by reducing the
expression of SP-D as exogenous steroids increase, rather than
reduce, SP-D expression in human lung [29]. It is more likely to
be reporting changes in permeability that result from suppres-
sion of inflammation. Thus SP-D is exquisitely more sensitive
in reporting the changes that result from the administration of
oral prednisolone than is lung function. It was difficult to
assess the effect of inhaled corticosteroids on serum SP-D level
in the present cohort as most subjects were taking this
medication.
The association of serum SP-D with COPD reported here arose
from a cross-sectional study. It will be important to assess
whether SP-D tracks with decline in lung function and
progression of emphysema, airways disease and systemic
features (such as BMI, fatigue, muscle wasting and systemic
inflammation) during the 3 yrs of follow-up of the ECLIPSE
cohort. If so, then serum SP-D offers a real prospect of a
biomarker that can report disease progression. Other studies
are needed to determine whether small molecules that reduce
inflammation and suppress SP-D can reduce exacerbations and
modify the decline in one or more of the indices that are
abnormal in individuals with COPD. If this is the case, then the
suppression of serum SP-D levels would provide an inter-
mediate measure of disease modification in COPD.
In summary, a large cohort of individuals with COPD and
smoking and nonsmoking controls have been used to show
that median serum SP-D levels are elevated, and predict
exacerbations, in individuals with COPD. These levels fall
following treatment with oral corticosteroids. Thus serum
SP-D may be useful as an intermediate measure in the
development of anti-inflammatory therapies for COPD.
CLINICAL TRIALS
This study is registered at ClinicalTrials.gov (trial numbers
NCT00292552 and NCT00379730).
STATEMENT OF INTEREST
Statements of interest for D.A. Lomas, E.K. Silverman, L.D. Edwards,
B.E. Miller, D.H. Horstman and R. Tal-Singer and the study itself can
be found at www.erj.ersjournals.com/misc/statements.dtl
ACKNOWLEDGEMENTS
The authors would like to express their thanks to all of the subjects
who took part in the present studies. They are grateful to R. Harding
(GlaxoSmithKline, London, UK) for help with study NCT00379730 and
E. Kurali (GlaxoSmithKline, King of Prussia, PA, USA) for exploratory
statistics. The quantitative computed tomography (CT) analysis was
performed by the Thoracic Imaging Group at the University of British
Columbia (Vancouver, BC, Canada) under the direction of H. Coxson,
and the CT scans were qualitatively assessed by N. Mu ller and
P. Nasute Fauerbach.
Principal investigators and centres participating in the Evaluation of
COPD Longitudinally to Identify Predictive Surrogate Endpoints study
(NCT00292552): Bulgaria: Y. Ivanov (Pleven), and K. Kostov (Sofia);
Canada: J. Bourbeau (Montreal, QC), M. Fitzgerald (Vancouver, BC),
P. Hernandez (Halifax, NS), K. Killian (Hamilton, ON), R. Levy
(Vancouver, BC), F. Maltais (Montreal, QC), and D. ODonnell
(Kingston, ON); Czech Republic: J. Krepelka (Prague); Denmark:
J. Vestbo (Hvidovre); the Netherlands: E. Wouters (Horn and
Maastricht); New Zealand: D. Quinn (Wellington); Norway: P. Bakke
(Bergen); Slovenia: M. Kosnik (Golnik); Spain: A. Agust (Palma de
Mallorca), and J. Sauleda (Palma de Mallorca); Ukraine: Y. Feschenko
(Kiev), V. Gavrisyuk (Kiev), N. Monogarova (Donetsk), and L. Yashina
(Kiev); UK: P. Calverley (Liverpool), D. Lomas (Cambridge),
W. MacNee (Edinburgh), D. Singh (Manchester), and J. Wedzicha
(London); and USA: A. Anzueto (San Antonio, TX), S. Braman
(Providence, RI), R. Casaburi (Torrance CA), B. Celli (Boston, MA),
G. Giessel (Richmond, VA), M. Gotfried (Phoenix, AZ), G. Greenwald
(Rancho Mirage, CA), N. Hanania (Houston, TX), D. Mahler (Lebanon,
NH), B. Make (Denver, CO), S. Rennard (Omaha, NE), C. Rochester
(New Haven, CT), P. Scanlon (Rochester, MN), D. Schuller (Omaha,
NE), F. Sciurba (Pittsburgh, PA), A. Sharafkhaneh (Houston, TX),
T. Siler (St Charles, MO), E. Silverman (Boston, MA), A. Wanner
(Miami, FL), R. Wise (Baltimore, MD), and R. ZuWallack (Hartford,
CT).
Steering committee: H. Coxson (Vancouver, Canada); L. Edwards
(GlaxoSmithKline, Research Triangle Park, NC, USA); K. Knobil (co-
chair; GlaxoSmithKline, Research Triangle Park, NC, USA); D. Lomas
(Cambridge, UK); W. MacNee (Edinburgh, UK); E. Silverman (Boston,
MA, USA); R. Tal-Singer (GlaxoSmithKline, King of Prussia, PA, USA);
J. Vestbo (co-chair; Hvidovre, Denmark); and J. Yates
(GlaxoSmithKline, Research Triangle Park, NC, USA).
Scientific committee: A. Agust (Barcelona, Spain); P. Calverley
(Liverpool, UK); B. Celli (Boston, MA, USA); C. Crim
(GlaxoSmithKline, Research Triangle Park, NC, USA); B. Miller
(GlaxoSmithKline, King of Prussia, PA, USA); W. MacNee (chair;
Edinburgh, UK); S. Rennard (Omaha, NE, USA); R. Tal-Singer
(GlaxoSmithKline, King of Prussia, PA, USA); E. Wouters (Horn,
Maastricht, the Netherlands); and J. Yates (GlaxoSmithKline, Research
Triangle Park, NC, USA).
Principal investigators and centres participating in the assessment of
oral corticosteroids in individuals with chronic obstructive pulmonary
disease (NCT00379730): E. Bateman (principal investigator; Cape
Town, South Africa); P. Elias (Mendoza, Argentina); F. Galleguillos
(Santiago, Chile); D. Quinn (Wellington, New Zealand); and
L. Vicherat (Santiago, Chile).
REFERENCES
1 Agusti AG. COPD, a multicomponent disease: implications for
management. Respir Med 2005; 99: 670682.
2 Celli BR. Chronic obstructive pulmonary disease phenotypes and
their clinical relevance. Proc Am Thorac Soc 2006; 3: 461466.
3 Rabe KF, Hurd S, Anzueto A, et al. Global strategy for the
diagnosis, management, and prevention of chronic obstructive
pulmonary disease: GOLD executive summary. Am J Respir Crit
Care Med 2007; 176: 532555.
4 Cazzola M, MacNee W, Martinez FJ, et al. Outcomes for COPD
pharmacological trials: from lung function to biomarkers. Eur
Respir J 2008; 31: 416469.
5 Kishore U, Greenhough TJ, Waters P, et al. Surfactant proteins
SP-A and SP-D: structure, function and receptors. Mol Immunol
2006; 43: 12931315.
6 Mori K, Kurihara N, Hayashida S, et al. The intrauterine
expression of surfactant protein D in the terminal airways of
human fetuses compared with surfactant protein A. Eur J Pediatr
2002; 161: 431434.
7 Kierstein S, Poulain FR, Cao Y, et al. Susceptibility to ozone-
induced airway inflammation is associated with decreased levels
of surfactant protein D. Respir Res 2006; 7: 85.
D.A. LOMAS ET AL. COPD AND SMOKING-RELATED DISORDERS
c
EUROPEAN RESPIRATORY JOURNAL VOLUME 34 NUMBER 1 101
8 Knudsen L, Ochs M, Mackay R, et al. Truncated recombinant
human SP-D attenuates emphysema and type II cell changes in
SP-D deficient mice. Respir Res 2007; 8: 70.
9 Leth-Larsen R, Nordenbaek C, Tornoe I, et al. Surfactant protein D
(SP-D) serum levels in patients with community-acquired pneu-
monia. Clin Immunol 2003; 108: 2937.
10 Miyata M, Sakuma F, Fukaya E, et al. Detection and monitoring of
methotrexate-associated lung injury using serum markers KL-6
and SP-D in rheumatoid arthritis. Intern Med 2002; 41: 467473.
11 Umetani K, Abe M, Kawabata K, et al. SP-D as a marker of
amiodarone-induced pulmonary toxicity, 2002; 41: 709712.
12 Ohnishi H, Yokoyama A, Kondo K, et al. Comparative study of
KL-6, surfactant protein-A, surfactant protein-D, and monocyte
chemoattractant protein-1 as serum markers for interstitial lung
diseases. Am J Respir Crit Care Med 2002; 165: 378381.
13 Krane M, Griese M. Surfactant protein D in serum from patients
with allergic bronchopulmonary aspergillosis. Eur Respir J 2003;
22: 592595.
14 Sims MW, Tal-Singer RM, Kierstein S, et al. Chronic
obstructive pulmonary disease and inhaled steroids alter surfac-
tant protein D (SP-D) levels: a cross-sectional study. Respir Res
2008; 9: 13.
15 Sin DD, Leung R, Gan WQ, et al. Circulating surfactant protein D
as a potential lung-specific biomarker of health outcomes in
COPD: a pilot study. BMC Pulm Med 2007; 7: 13.
16 Vestbo J, Anderson W, Coxson HO, et al. Evaluation of COPD
Longitudinally to Identify Predictive Surrogate End-points
(ECLIPSE). Eur Respir J 2008; 31: 869873.
17 Lomas DA, Silverman EK, Edwards LD, et al. Evaluation of serum
CC-16 as a biomarker for chronic obstructive pulmonary disease in
the ECLIPSE cohort. Thorax 2008; 63: 10581063.
18 American Thoracic Society. Standardization of spirometry. Am J
Respir Crit Care Med 1994; 152: 11071136.
19 Medical Research Council. Definition and classification of chronic
bronchitis for clinical and epidemiological purposes. Lancet 1965; i:
775779.
20 Bland JM, Altman DG. Statistical methods for assessing agreement
between two methods of clinical measurement. Lancet 1986; i:
307310.
21 Srensen GL, Hjelmborg JB, Kyvik KO, et al. Genetic and
environmental influences of surfactant protein D serum levels.
Am J Physiol Lung Cell Mol Physiol 2006; 290: L1010L1017.
22 Sin DD, Man S-FP, Marciniuk DD, et al. The effects of fluticasone
with or without salmeterol on systemic biomarkers of inflamma-
tion in COPD. Am J Respir Crit Care Med 2008; 177: 12071214.
23 Cao Y, Grous M, Scanlon ST, et al. The innate immune molecule
surfactant protein (SP)-D is upregulated in the lung following
cigarette smoke (CS) exposure in a murine model. Am J Respir Crit
Care Med 2004; 169: A832.
24 Noah TL, Murphy PC, Alink JJ, et al. Bronchoalveolar lavage fluid
surfactant protein-A and surfactant protein-D are inversely related
to inflammation in early cystic fibrosis. Am J Respir Crit Care Med
2003; 168: 685691.
25 Hurst JR, Donaldson GC, Perera WR, et al. Use of plasma
biomarkers at exacerbations of chronic obstructive pulmonary
disease. Am J Respir Crit Care Med 2006; 174: 867874.
26 Bozinovski S, Hutchinson A, Thompson M, et al. Serum amyloid a
is a biomarker of acute exacerbations of chronic obstructive
pulmonary disease. Am J Respir Crit Care Med 2008; 177: 269278.
27 Papi A, Bellettato CM, Braccioni F, et al. Infections and airway
inflammation in chronic obstructive pulmonary disease severe
exacerbations. Am J Respir Crit Care Med 2006; 173: 11141121.
28 Celli BR, Barnes PJ. Exacerbations of chronic obstructive pulmon-
ary disease. Eur Respir J 2007; 29: 12241238.
29 Wang JY, Yeh TF, Lin YC, et al. Measurement of pulmonary status
and surfactant protein levels during dexamethasone treatment of
neonatal respiratory distress syndrome. Thorax 1996; 51: 907913.
COPD AND SMOKING-RELATED DISORDERS D.A. LOMAS ET AL.
102 VOLUME 34 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL