Acute Asthma in Children
Relationships among CD14 and CC16 Genotypes, Plasma Levels, and Severity
Andrew C. Martin, Ingrid A. Laing, Siew-Kim Khoo, Guicheng Zhang, Kristina Rueter, Laurel Teoh,
Shahir Taheri†, Catherine M. Hayden, Gary C. Geelhoed, Jack Goldblatt, and Peter N. LeSouef
School of Paediatrics and Child Health, University of Western Australia, Perth, Western Australia, Australia
Rationale: The majority of previous studies investigating asthma extensively studied in adults and children (9–12). Differences in
genetics have focused on cohorts with stable disease and have not gene expression have been identiﬁed among individuals with
defined mechanisms important during acute asthma. CD14 and stable asthma, yet dysregulation of pro- or antiinﬂammatory
CC16 each play a key role in biologically important inflammatory processes is likely to have the most critical inﬂuence during the
pathways and the gene of each has a functional promoter-region early stages of an acute asthma attack, when the loss of control
polymorphism. of inﬂammation could be expected to be maximal. CD14 is a
Objectives: This study was designed to determine the influence of key component of the innate immune system and its gene is
these polymorphisms on plasma levels of their products and clinical located on chromosome 5q31.1. It is expressed on monocytes
disease during acute asthma. We hypothesized that genotype-
and macrophages, functions as a receptor for LPS (13), and
related differences in CD14 and CC16 production would be more
exists in a membrane-bound form and a soluble form (sCD14).
marked during acute asthma and related to disease severity.
CD14 plays a critical role in determining the balance of Th1:Th2
Methods: We studied 148 children on presentation with acute
asthma and again in convalescence. CD14 C-159T and CC16 A38G cytokines, with activation promoting the release of interleukin
genotypes were determined, and plasma levels of soluble CD14 12 (IL-12) and deviation of immune responses toward an antivi-
(sCD14) and CC16 were measured at both times. ral T-helper type 1 (Th1) response (14). Levels of sCD14 have
Measurements and Main Results: During acute asthma, plasma sCD14 been noted to be higher in individuals with asthma than in those
levels were higher for the whole group (p 0.003), but increases without asthma and also higher during acute asthma exacerba-
were only in subjects with CD14 159TT (p 0.003) and 159CT tions as compared with convalescence (15). A promoter poly-
(p 0.004), and not in those with 159CC. Plasma CC16 levels were morphism (C-159T) was identiﬁed, and the 159C allele associ-
also elevated acutely for the whole group (p 0.013), but only in ated with lower levels of sCD14 in an unselected population of
those with CC16 38GG (p 0.043) and 38AG (p 0.014), and not children (11), but the role of this polymorphism during acute
in those with CC16 38AA. Subjects with CD14 159CC and CC16 asthma is unknown. Clara cell secretory protein (CC16) is pro-
38AA were more likely to have moderate or severe acute asthma. duced by bronchiolar, nonciliated epithelial cells (5) and is the
Conclusions: Plasma levels of sCD14 and CC16 were higher during acute most abundant protein secreted into the airway (16), functioning
asthma in the subjects. Those with CD14 159CC and CC16 38AA as an immunosuppressive and antiinﬂammatory agent (5, 17).
had no change in sCD14 and CC16 levels and more severe asthma. Although CC16 is produced in the airway, serum levels mirror
those in the lung and individuals with asthma have lower circulat-
Keywords: asthma; children; single nucleotide polymorphism
ing levels than those without asthma (18). The gene for CC16
Acute asthma is the most common diagnosis in children admitted is located on chromosome 11q13 and has a functional promoter-
to hospitals in Western society and is characterized by acute region polymorphism (A38G) (8, 9). The 38G allele was associ-
episodes of obstruction related to loss of control of airway in- ated with a decreased risk of developing childhood asthma (9)
ﬂammation mostly in response to a viral respiratory-tract infec- and increased gene expression levels in vitro, but the role of this
tion (1). Many genetic variations associated with asthma and allele in acute asthma is unknown.
asthma phenotypes have been reported (2–4). However, these Given the rapid onset of inﬂammation in an acute asthma
investigations have focused on asthma phenotypes in cohorts attack, we hypothesized that genotype-related differences in pro-
with stable disease and no studies have systematically evaluated duction of CD14 and CC16 would be more marked during acute
the inﬂuence of genetic differences in acute asthma. asthma than convalescence and that these differences would be
CD14 and CC16 are biologically important in clearly deﬁned associated with altered phenotypes.
immunologic and inﬂammatory pathways (5, 6). Single nucleo- Some of the results of this study have been previously re-
tide polymorphisms (SNPs) in the promoter regions of their ported in the form of abstracts (19, 20).
genes alter the amount of expressed protein (7, 8) and have been
(Received in original form September 3, 2005; accepted in final form December 27, 2005) A total of 148 children, aged 2 to 16 yr, presenting with acute asthma to
the Emergency Department at Princess Margaret Hospital for Children,
Supported by grants from the National Health and Medical Research Council of Perth, Western Australia, were recruited between July 2002 and
Australia, Asthma Foundation of Western Australia, and the West Australian Insti-
September 2004. An acute asthma attack was diagnosed by the emer-
tute of Medical Research.
gency department physician based on the presence of wheezing with
Dr. Shahir Taheri is deceased. increased difﬁculty of breathing. Parents of all participants gave
Correspondence and requests for reprints should be addressed to Andrew Martin, informed consent and the Princess Margaret Hospital Ethics Committee
M.B.B.S., M.R.C.P., Princess Margaret Hospital for Children, Roberts Road, Subiaco, approved the study.
Western Australia 6008, Australia. E-mail: email@example.com The severity of the acute asthma attack at presentation was deter-
This article has an online supplement, which is accessible from this issue’s table mined using a previously validated scoring system with a possible score
of contents at www.atsjournals.org in the range 5 to 15 determined for each subject: 5 to 7, mild; 8 to 11,
Am J Respir Crit Care Med Vol 173. pp 617–622, 2006
moderate; 12 to 15, severe (21). Additional details of the scoring system
Originally Published in Press as DOI: 10.1164/rccm.200509-1367OC on December 30, 2005 are provided in the online supplement. All children received inhaled
Internet address: www.atsjournals.org salbutamol and ipratropium bromide at 20-min intervals for the ﬁrst
618 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 173 2006
hour, prednisolone 1 mg/kg orally, and if saturations were less than or The C allele frequency for CD14 C-159T was 45.3% and A
equal to 94%, supplemental oxygen was administered. The pattern allele frequency for CC16 A38G was 31.9%, both similar to
of asthma severity was also assessed according to National Asthma frequencies from an unselected population in the same city (25,
Council of Australia guidelines to determine whether children suffered
from infrequent episodic, frequent episodic, or persistent asthma
26). The genotype distributions did not deviate from Hardy-
exacerbations (22). Weinberg equilibrium. Table 2 shows the genotype frequencies.
Samples sCD14 and CC16: Acute versus Convalescent Plasma Levels
Peripheral blood samples were obtained as soon as possible after the Paired acute and convalescent plasma levels were available for 94
ﬁrst dose of prednisolone and always within 24 h of presentation to children who were representative of the cohort. During the acute
the hospital. A further blood sample was obtained from those who period, GM levels of both sCD14 and CC16 were higher than during
were clinically well and able to return at least 6 wk after the acute convalescence: CD14, 3.03 g/ml (95% CI, 2.80–3.28 g/ml)
attack. Buffy coat and plasma were separated and stored at 80 C. At
versus 2.61 g/ml (95% CI, 2.42–2.82 g/ml), p 0.003; CC16,
presentation, a per-nasal aspirate was obtained for detection of common
viral respiratory pathogens and a skin-prick test for 11 common aller- 2.23 ng/ml (95% CI, 1.89–2.64 ng/ml) versus 1.78 ng/ml (95%
gens was done. A positive reaction was deﬁned as a wheal size that CI, 1.48–2.14 ng/ml), p 0.013 (Figure 1).
was larger than the negative control and greater than or equal to 3 mm
in diameter. Atopy was deﬁned as at least one positive skin-prick test. sCD14 and CC16 versus the Corresponding Genotypes
During the acute episodes of asthma (n 148), plasma levels
of sCD14 and CC16 were signiﬁcantly related to genotype for
Genomic DNA was extracted by standard techniques (23) and the CD14 CD14 C-159T and CC16 A38G, respectively. Mean plasma
C-159T and CC16 A38G genotypes were determined by restriction
sCD14 levels were highest in subjects with CD14 159TT (GM,
digestion of polymerase chain reaction products using the restriction
enzymes AvaII and Sau96I (Promega, Madison, WI), respectively (9, 11). 3.6 g/ml; 95% CI, 3.3–4.0 g/ml), followed by heterozygotes
(GM, 3.2 g/ml; 95% CI, 2.9–3.5 g/ml), and lowest for those
Plasma Assays with CD14 159CC (GM, 2.5 g/ml; 95% CI, 2.1–2.9 g/ml),
Plasma levels of sCD14 and CC16 were determined using commercially p 0.001. When levels during the acute attacks were compared
available ELISA kits (R&D Systems, Minneapolis, MN; and Bio- with levels during convalescence (n 94), higher plasma levels
Vendor, Brno, Czech Republic; respectively). of sCD14 were present during the acute attack in those with
159CT and 159TT (p 0.004 and p 0.003, respectively),
but no difference was present in those with CD14 159CC
Because plasma levels of sCD14 and CC16 were both positively skewed, (Figure 2). Similarly, during the acute attack, mean plasma CC16
geometric means (GM) and 95% conﬁdence intervals (CI) were calcu- level was highest in those with 38GG (GM, 2.4 ng/ml; 95% CI,
lated after applying a logarithmic transformation that resulted in a
normal distribution. As the distribution of asthma severity scores was
2.0–2.9), intermediate in heterozygotes (GM, 2.0 ng/ml; 95% CI,
approximately normal, parametric statistics were employed. The differ- 1.7–2.5), and lowest in those with 38AA (GM, 1.2 ng/ml; 95%
ences in plasma levels of sCD14 and CC16 between acute asthma and CI, 0.8–1.8 ng/ml), p 0.025. When levels during acute attacks
convalescence were compared by paired-sample t tests. Analysis of were compared with levels during convalescence (n 94), higher
variance was used to compare the difference in plasma levels of sCD14 plasma levels of CC16 were present during the acute attack in
and CC16 and asthma severity scores between the genotypes, with a those with 38GG and 38AG (p 0.014 and p 0.043, respec-
polynomial linear analysis for trend. Bivariate Pearson correlation was
employed to explore the association between asthma severity scores
tively), whereas in 38AA homozygotes, CC16 levels were actu-
and plasma sCD14 and CC16. To estimate the odds ratios (OR) for ally lower during the acute attacks than during convalescence,
moderate or severe acute asthma attacks in children with CD14 -159CC although this difference was not statistically signiﬁcant (p
and CC16 38AA, logistic regression models were applied. All statistics 0.088; Figure 3). For CD14 C-159T and CC16 A38G, there was
were analyzed using SPSS (SPSS for Windows, Release 11; SPSS, a signiﬁcant linear effect for each additional C or A allele, respec-
Chicago, IL) (24). tively, on plasma sCD14 and CC16 levels (p 0.001 and p
0.007, respectively). During convalescence, no signiﬁcant differ-
RESULTS ence in plasma levels of sCD14 and CC16 was found between
Characteristics of the children recruited for the study are shown the genotype groups for the CD14 C-159T and CC16 A38G
in Table 1. polymorphisms. We also explored the impact of viral infection
and atopy on plasma sCD14 and CC16 levels and found that
neither had a signiﬁcant effect. (Comparing subjects with and
TABLE 1. CHARACTERISTICS OF THE STUDY POPULATION without viral infection, p 0.89 and p 0.59 for sCD14 and
CC16 levels, respectively; and for atopy, p 0.58 and p 0.80,
Characteristic Subjects (n 148) respectively.)
Mean age, yr SD 6.6 3.6
Males/females 89/59 Asthma Severity in Relation to CD14 C-159T and CC16 A38G
Ethnicity, % white 92.3 Genotypes and Plasma Levels
Preceding symptoms of URTI, n (%) 120 (81.1)
In the 148 children assessed during acute attacks, children with
Virus isolated, n* (%) 89 (75.4)
Oxygen saturation on arrival, % SD 93.8 3.6 the CD14 159CC and CC16 38AA genotypes had higher mean
Mean asthma severity score SD 9.5 2.4 asthma severity scores compared with the other genotypes
Mild asthma, n (%) 32 (21.6)
Moderate asthma, n (%) 86 (58.1)
Severe asthma, n (%) 30 (20.3)
Infrequent episodic asthma, n (%) 98 (66.2) TABLE 2. CD14 AND CC16 GENOTYPE FREQUENCIES
Frequent episodic asthma, n (%) 23 (15.5)
Persistent asthma, n (%) 27 (18.3) CD14 C-159T CC CT TT
Atopy, n* (%) 94 (79.3) n, % 36 (24.3) 62 (41.9) 50 (33.8)
CC16 A38G AA AG GG
Definition of abbreviation: URTI upper respiratory tract infection.
n, % 13 (8.8) 68 (45.9) 67 (45.3)
* Test completed in only 118 subjects.
Martin, Laing, Zhang, et al.: Genetics and Acute Asthma in Children 619
Figure 1. Paired plasma levels of soluble CD14 (sCD14)
and CC16: acute versus convalescence (n 94).
(CD14 C-159T, CC 10.1 vs. CT 9.0 vs. TT 9.7, p 0.06; and clude a larger panel of genes, examining both individual SNPs
CC16 A38G, AA 9.9 vs. AG 9.6 vs. GG 9.3, p 0.76). Logistic and haplotype patterns.
regression analysis was undertaken to further investigate the As previously noted, CD14 is known to play an important
association between asthma severity and these genotypes. After role in the immune system and plasma levels were increased
adjustment for age and sex, children with CD14 159CC were with acute asthma. However, the precise physiologic role of
more likely to have an asthma severity score indicating a moder- sCD14 is still unclear, as it may be beneﬁcial as a scavenger to
ate or severe attack (odds ratio [OR], 3.7; 95% CI, 1.04–13.2; neutralize circulating LPS, or its absence on endothelial cells
p 0.043) compared with those with 159CT and 159TT. that do not express membrane-bound CD14 may allow LPS to
An inverse correlation (coefﬁcient, 0.17; p 0.05) was found produce an aggressive and harmful proinﬂammatory cytokine
between asthma severity scores and the log value of sCD14 response. Increased serum levels of sCD14 have been reported
during acute asthma attacks. Subjects with CC16 38AA also in severe acute systemic conditions, such as gram-negative septi-
tended to have an asthma severity score in the moderate or cemia, polytrauma, burns, and acute respiratory distress syn-
severe range, but the difference from those with 38AG and drome and have been associated with increased morbidity and
38GG were not signiﬁcant (OR, 3.7; 95% CI, 0.45–30; p 0.231). mortality (27–29). Studies of IC14, a CD14-speciﬁc monoclonal
This same pattern for both CD14 and CC16 was found when antibody, showed the potential of this treatment to limit the
only the subjects with paired samples (n 94) were analyzed. excessive systemic inﬂammatory response in subjects with severe
We also investigated the possibility of an additive or syner- sepsis (30). Consistent with these reports, the current study found
gistic effect of the CD14 159CC and CC16 38AA genotypes that during acute asthma attacks in children, plasma sCD14
on the severity of an acute asthma attack, but found no signiﬁcant levels were inversely correlated with severity, suggesting a pro-
gene–gene interaction. tective role for sCD14. Plasma sCD14 levels have been directly
correlated with IFN- and inversely correlated with IL-4 (11),
DISCUSSION suggesting that in children with acute asthma, the beneﬁcial
The present study is the ﬁrst to our knowledge to use genetics effect of greater CD14 activity may result from increased Th1
to investigate the mechanisms of asthma in children during acute and decreased Th2 responses. A relative predominance of Th1
exacerbation. This approach clearly has the potential to elucidate over Th2 cytokines assists in the elimination of viral infections
mechanisms underlying acute asthma, as transcription of genes (31), thus lower levels of sCD14 may allow ongoing viral replica-
involved in asthmatic inﬂammation is likely to be maximal during tion and inﬂammation.
the acute exacerbation, and therefore, allele-speciﬁc differences Acute asthma attacks are associated with a marked increase
should be most evident. in airway inﬂammation and this was reﬂected by the ﬁnding of
In the present study, we have investigated two key agents mean CC16 plasma levels being 95.9% higher during the acute
involved in inﬂammation and demonstrated clear patterns relat- attacks compared with convalescence. Plasma levels of CC16
ing their genotypes to both asthma severity and plasma levels. are reported to reﬂect CC16 levels in bronchoalveolar lavage in
These patterns were present during the acute attacks and not healthy adults (16). This study is the ﬁrst to demonstrate altered
detectable on recovery. Speciﬁcally, we found that levels of both circulating CC16 levels in subjects during an acute attack. The
CD14 and CC16 were increased during the acute episode, but only other study comparing acute and convalescent plasma CC16
that the increases were seen with one but not the other allele levels did not ﬁnd a difference, but compared paired plasma
of each of the polymorphisms. These ﬁndings were plausible CC16 levels in only 10 adults with asthma (18).
and linked to clinical status, as, for both CD14 and CC16, the This study demonstrated that genotype-speciﬁc differences
allele for which there was no increase in plasma level was associ- in plasma levels of sCD14 and CC16 during acute exacerbation
ated with more severe acute asthma. Because these ﬁndings were even more pronounced than during convalescence. For
could not be made studying individuals with stable asthma, they CD14, subjects with 159TT and 159CT genotypes had 42
make an important contribution to current knowledge of the and 31% higher plasma sCD14 levels, respectively, compared
mechanisms of asthma. The two genes we studied were speciﬁ- with 10% lower levels in those with 159CC. The ﬁnding that
cally selected because of their involvement in inﬂammatory path- during acute asthma 159TT subjects had the highest sCD14
ways, but studies on similar cohorts should be extended to in- levels, heterozygotes intermediate, and 159CC the lowest
620 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 173 2006
Figure 2. Relationship between plasma lev-
els of sCD14 and genotypes of CD14 C-159T.
levels was consistent in direction with those from a normal popu- greatly exceeds production during acute asthma. Subjects with
lation of American school children (11). However, in contrast to asthma with the 38A allele may be unable to increase levels of
the study by Baldini and colleagues, which suggested a dominant CC16 in the airway at the time of viral infections when protection
model, we found a linear effect, with each additional T allele from excessive airway inﬂammation becomes most critical. This
having an additive effect on plasma sCD14 levels. Interestingly, may result in 38AA subjects experiencing more severe acute
this linear effect was only present during the acute asthma exac- asthma due to lower levels of this endogenous antiinﬂammatory
erbation, suggesting that gene expression at this time is likely agent.
to be exaggerated. Levels of sCD14 were similar for the different Those subjects who were either CD14 159CC or CC16
genotypes in convalescence, further demonstrating that this 38AA were over three times more likely to have moderate or
pathogenetic process could only have been identiﬁed by studying severe attacks of acute asthma compared with the other geno-
children during acute attacks. Our ﬁndings were also consistent types. A potential mechanism in determining severity of an acute
with functional studies that showed that the T allele produced attack is impaired ability to increase sCD14 and CC16. The
32% greater activity than the C allele (7). effect of CD14 C-159T on asthma severity was partly dependent
For CC16, subjects with 38GG and 38AG genotypes had on plasma sCD14 levels, with higher levels associated with a
134% and 80% higher plasma CC16 levels, respectively, com- milder asthma attack, possibly because of increased Th1 cytokine
pared with 38AA subjects, who paradoxically had plasma CC16 release. Although differences for CC16 did not reach statistical
levels that were 33% lower. These genotype-speciﬁc differences signiﬁcance, mildly affected subjects tended to have the highest
in plasma levels were also consistent in direction with the ﬁndings CC16 levels and severely affected the lowest. However, as CC16
from a case control study of Australian children (25). However, levels in plasma are approximately 10,000 times lower than in
in addition to having the lowest levels of CC16 during an acute bronchoalveolar lavage (32), any changes seen in the plasma are
asthma attack, subjects with 38AA appeared to have had a de- likely to be substantially more pronounced in the airway.
crease in plasma CC16 levels when the degree of airway inﬂam- Although this study population was relatively large compared
mation was maximal. This difference may reﬂect decreased gene with other studies of children with acute asthma, the number of
expression by the A allele or that the consumption of CC16 subjects with some genotypes, such as CC16 38AA, was relatively
Martin, Laing, Zhang, et al.: Genetics and Acute Asthma in Children 621
Figure 3. Relationship between plasma lev-
els of CC16 and genotypes of CC16 A38G.
small compared with other genetic association studies. However, effect on the polymorphisms studied. Furthermore, the highly
this study differed from previous studies in that the stimulation signiﬁcant ﬁndings of genotype-speciﬁc variation in acute plasma
of inﬂammatory pathways was likely to have been near maximal levels of sCD14 and CC16 suggested that, even if steroids inﬂu-
and, for the genes assessed, the genes’ own product was an enced the absolute level of gene product, genotype differences
important primary outcome variable. Also, genotype-speciﬁc still play a major role in determining altered levels of gene
plasma levels of sCD14 and CC16 were consistent with previous product between individuals and asthma severity. Ethnicity is
studies, as well as being internally consistent between the geno- known to inﬂuence the genotype frequency for both CD14
types, and these consistencies strongly support the validity of C-159T and CC16 A38G (35). However, as more than 90% of
the ﬁndings. Thus, the striking and consistent associations found, our population were white Australians, of families originally
despite the relatively small numbers of subjects, vindicate this from Europe, the low frequency of other ethnic origins meant
novel approach of studying genetic associations at a time when that we were unable to investigate the effect of ethnicity in this
the loss of control of inﬂammation could be expected to be study. In studying genetic associations, consideration must be
maximal. Such an approach has the potential to allow the identi- also given to the presence of multiple SNPs per gene and to
ﬁcation of important genetic associations in relatively small functional haplotypes. In the present study, only one SNP was
cohorts. examined for each gene, but CD14 C-159T and CC16 A38G are
Other potential limitations should be considered. Although considered the most important CD14 and CC16 SNPs, as they
children were studied early in the course of the acute attack, in are the only ones shown to have functional signiﬁcance.
all cases blood samples were obtained after the ﬁrst dose of In summary, this study of children during acute asthma at-
prednisolone. Exogenous steroids can promote down-regulation tacks identiﬁed signiﬁcant relationships between two common
of sCD14 and up-regulation of CC16 (33, 34), but there is no promoter SNPs in the CD14 and CC16 genes and genotype-
evidence to suggest that steroids would have an allele-speciﬁc speciﬁc differences in their gene products’ plasma levels, which
622 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 173 2006
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Conflict of Interest Statement : None of the authors have a financial relationship
J, Le Souef PN. Relationship between CD14C–159T and soluble CD14
with a commercial entity that has an interest in the subject of this manuscript.
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Acknowledgment : The authors thank the children and families who participated (abstract).
in this study and acknowledge the technical assistance of Jenny Tizard. 20. Martin AC, Laing IA, Taheri S, Teoh L, Rueter K, Zhang G, Khoo S,
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