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School of Paediatrics and Child Health, University of Western Australia, Perth, Western Australia, Australia
ABSTRACT
Rationale: The majority of previous studies investigating asthma genetics have focused on cohorts with stable disease and have not defined mechanisms important during acute asthma. CD14 and CC16 each play a key role in biologically important inflammatory pathways and the gene of each has a functional promoter-region polymorphism.
Objectives: This study was designed to determine the influence of these polymorphisms on plasma levels of their products and clinical disease during acute asthma. We hypothesized that genotype-related differences in CD14 and CC16 production would be more marked during acute asthma and related to disease severity.
Methods: We studied 148 children on presentation with acute asthma and again in convalescence. CD14 C-159T and CC16 A38G genotypes were determined, and plasma levels of soluble CD14 (sCD14) and CC16 were measured at both times.
Measurements and Main Results: During acute asthma, plasma sCD14 levels were higher for the whole group (p = 0.003), but increases were only in subjects with CD14 –159TT (p = 0.003) and –159CT (p = 0.004), and not in those with –159CC. Plasma CC16 levels were also elevated acutely for the whole group (p = 0.013), but only in those with CC16 38GG (p = 0.043) and 38AG (p = 0.014), and not in those with CC16 38AA. Subjects with CD14 –159CC and CC16 38AA were more likely to have moderate or severe acute asthma.
Conclusions: Plasma levels of sCD14 and CC16 were higher during acute asthma in the subjects. Those with CD14 –159CC and CC16 38AA had no change in sCD14 and CC16 levels and more severe asthma.
Key Words: asthma children single nucleotide polymorphism
Acute asthma is the most common diagnosis in children admitted to hospitals in Western society and is characterized by acute episodes of obstruction related to loss of control of airway inflammation mostly in response to a viral respiratory-tract infection (1). Many genetic variations associated with asthma and asthma phenotypes have been reported (2–4). However, these investigations have focused on asthma phenotypes in cohorts with stable disease and no studies have systematically evaluated the influence of genetic differences in acute asthma.
CD14 and CC16 are biologically important in clearly defined immunologic and inflammatory pathways (5, 6). Single nucleotide polymorphisms (SNPs) in the promoter regions of their genes alter the amount of expressed protein (7, 8) and have been extensively studied in adults and children (9–12). Differences in gene expression have been identified among individuals with stable asthma, yet dysregulation of pro- or antiinflammatory processes is likely to have the most critical influence during the early stages of an acute asthma attack, when the loss of control of inflammation could be expected to be maximal. CD14 is a key component of the innate immune system and its gene is located on chromosome 5q31.1. It is expressed on monocytes and macrophages, functions as a receptor for LPS (13), and exists in a membrane-bound form and a soluble form (sCD14). CD14 plays a critical role in determining the balance of Th1:Th2 cytokines, with activation promoting the release of interleukin 12 (IL-12) and deviation of immune responses toward an antiviral T-helper type 1 (Th1) response (14). Levels of sCD14 have been noted to be higher in individuals with asthma than in those without asthma and also higher during acute asthma exacerbations as compared with convalescence (15). A promoter polymorphism (C-159T) was identified, and the –159C allele associated with lower levels of sCD14 in an unselected population of children (11), but the role of this polymorphism during acute asthma is unknown. Clara cell secretory protein (CC16) is produced by bronchiolar, nonciliated epithelial cells (5) and is the most abundant protein secreted into the airway (16), functioning as an immunosuppressive and antiinflammatory agent (5, 17). Although CC16 is produced in the airway, serum levels mirror those in the lung and individuals with asthma have lower circulating levels than those without asthma (18). The gene for CC16 is located on chromosome 11q13 and has a functional promoter-region polymorphism (A38G) (8, 9). The 38G allele was associated with a decreased risk of developing childhood asthma (9) and increased gene expression levels in vitro, but the role of this allele in acute asthma is unknown.
Given the rapid onset of inflammation in an acute asthma attack, we hypothesized that genotype-related differences in production of CD14 and CC16 would be more marked during acute asthma than convalescence and that these differences would be associated with altered phenotypes.
Some of the results of this study have been previously reported in the form of abstracts (19, 20).
METHODS
Subjects
A total of 148 children, aged 2 to 16 yr, presenting with acute asthma to the Emergency Department at Princess Margaret Hospital for Children, Perth, Western Australia, were recruited between July 2002 and September 2004. An acute asthma attack was diagnosed by the emergency department physician based on the presence of wheezing with increased difficulty of breathing. Parents of all participants gave informed consent and the Princess Margaret Hospital Ethics Committee approved the study.
The severity of the acute asthma attack at presentation was determined using a previously validated scoring system with a possible score in the range 5 to 15 determined for each subject: 5 to 7, mild; 8 to 11, moderate; 12 to 15, severe (21). Additional details of the scoring system are provided in the online supplement. All children received inhaled salbutamol and ipratropium bromide at 20-min intervals for the first hour, prednisolone 1 mg/kg orally, and if saturations were less than or equal to 94%, supplemental oxygen was administered. The pattern of asthma severity was also assessed according to National Asthma Council of Australia guidelines to determine whether children suffered from infrequent episodic, frequent episodic, or persistent asthma exacerbations (22).
Samples
Peripheral blood samples were obtained as soon as possible after the first dose of prednisolone and always within 24 h of presentation to the hospital. A further blood sample was obtained from those who were clinically well and able to return at least 6 wk after the acute attack. Buffy coat and plasma were separated and stored at –80°C. At presentation, a per-nasal aspirate was obtained for detection of common viral respiratory pathogens and a skin-prick test for 11 common allergens was done. A positive reaction was defined as a wheal size that 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.
Genotyping
Genomic DNA was extracted by standard techniques (23) and the CD14 C-159T and CC16 A38G genotypes were determined by restriction digestion of polymerase chain reaction products using the restriction enzymes AvaII and Sau96I (Promega, Madison, WI), respectively (9, 11).
Plasma Assays
Plasma levels of sCD14 and CC16 were determined using commercially available ELISA kits (R&D Systems, Minneapolis, MN; and BioVendor, Brno, Czech Republic; respectively).
Statistical Analysis
Because plasma levels of sCD14 and CC16 were both positively skewed, geometric means (GM) and 95% confidence intervals (CI) were calculated after applying a logarithmic transformation that resulted in a normal distribution. As the distribution of asthma severity scores was approximately normal, parametric statistics were employed. The differences in plasma levels of sCD14 and CC16 between acute asthma and convalescence were compared by paired-sample t tests. Analysis of variance was used to compare the difference in plasma levels of sCD14 and CC16 and asthma severity scores between the genotypes, with a polynomial linear analysis for trend. Bivariate Pearson correlation was employed to explore the association between asthma severity scores and plasma sCD14 and CC16. To estimate the odds ratios (OR) for moderate or severe acute asthma attacks in children with CD14 -159CC and CC16 38AA, logistic regression models were applied. All statistics were analyzed using SPSS (SPSS for Windows, Release 11; SPSS, Chicago, IL) (24).
RESULTS
Characteristics of the children recruited for the study are shown in Table 1.
The C allele frequency for CD14 C-159T was 45.3% and A allele frequency for CC16 A38G was 31.9%, both similar to frequencies from an unselected population in the same city (25, 26). The genotype distributions did not deviate from Hardy-Weinberg equilibrium. Table 2 shows the genotype frequencies.
sCD14 and CC16: Acute versus Convalescent Plasma Levels
Paired acute and convalescent plasma levels were available for 94 children who were representative of the cohort. During the acute period, GM levels of both sCD14 and CC16 were higher than during convalescence: CD14, 3.03 μg/ml (95% CI, 2.80–3.28 μg/ml) versus 2.61 μg/ml (95% CI, 2.42–2.82 μg/ml), p = 0.003; CC16, 2.23 ng/ml (95% CI, 1.89–2.64 ng/ml) versus 1.78 ng/ml (95% CI, 1.48–2.14 ng/ml), p = 0.013 (Figure 1).
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 CD14 C-159T and CC16 A38G, respectively. Mean plasma sCD14 levels were highest in subjects with CD14 –159TT (GM, 3.6 μg/ml; 95% CI, 3.3–4.0 μg/ml), followed by heterozygotes (GM, 3.2 μg/ml; 95% CI, 2.9–3.5 μg/ml), and lowest for those with CD14 –159CC (GM, 2.5 μg/ml; 95% CI, 2.1–2.9 μg/ml), p < 0.001. When levels during the acute attacks were compared with levels during convalescence (n = 94), higher plasma levels 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 (Figure 2). Similarly, during the acute attack, mean plasma CC16 level was highest in those with 38GG (GM, 2.4 ng/ml; 95% CI, 2.0–2.9), intermediate in heterozygotes (GM, 2.0 ng/ml; 95% CI, 1.7–2.5), and lowest in those with 38AA (GM, 1.2 ng/ml; 95% CI, 0.8–1.8 ng/ml), p = 0.025. When levels during acute attacks were compared with levels during convalescence (n = 94), higher plasma levels of CC16 were present during the acute attack in those with 38GG and 38AG (p = 0.014 and p = 0.043, respectively), whereas in 38AA homozygotes, CC16 levels were actually lower during the acute attacks than during convalescence, although this difference was not statistically significant (p = 0.088; Figure 3). For CD14 C-159T and CC16 A38G, there was a significant linear effect for each additional C or A allele, respectively, on plasma sCD14 and CC16 levels (p < 0.001 and p = 0.007, respectively). During convalescence, no significant difference in plasma levels of sCD14 and CC16 was found between the genotype groups for the CD14 C-159T and CC16 A38G 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 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, respectively.)
Asthma Severity in Relation to CD14 C-159T and CC16 A38G Genotypes and Plasma Levels
In the 148 children assessed during acute attacks, children with the CD14 –159CC and CC16 38AA genotypes had higher mean asthma severity scores compared with the other genotypes (CD14 C-159T, CC 10.1 vs. CT 9.0 vs. TT 9.7, p = 0.06; and CC16 A38G, AA 9.9 vs. AG 9.6 vs. GG 9.3, p = 0.76). Logistic regression analysis was undertaken to further investigate the association between asthma severity and these genotypes. After adjustment for age and sex, children with CD14 –159CC were more likely to have an asthma severity score indicating a moderate or severe attack (odds ratio [OR], 3.7; 95% CI, 1.04–13.2; p = 0.043) compared with those with –159CT and –159TT. An inverse correlation (coefficient, –0.17; p < 0.05) was found between asthma severity scores and the log value of sCD14 during acute asthma attacks. Subjects with CC16 38AA also tended to have an asthma severity score in the moderate or severe range, but the difference from those with 38AG and 38GG were not significant (OR, 3.7; 95% CI, 0.45–30; p = 0.231). This same pattern for both CD14 and CC16 was found when only the subjects with paired samples (n = 94) were analyzed.
We also investigated the possibility of an additive or synergistic effect of the CD14 –159CC and CC16 38AA genotypes on the severity of an acute asthma attack, but found no significant gene–gene interaction.
DISCUSSION
The present study is the first to our knowledge to use genetics to investigate the mechanisms of asthma in children during acute exacerbation. This approach clearly has the potential to elucidate mechanisms underlying acute asthma, as transcription of genes involved in asthmatic inflammation is likely to be maximal during the acute exacerbation, and therefore, allele-specific differences should be most evident.
In the present study, we have investigated two key agents involved in inflammation and demonstrated clear patterns relating their genotypes to both asthma severity and plasma levels. These patterns were present during the acute attacks and not detectable on recovery. Specifically, we found that levels of both CD14 and CC16 were increased during the acute episode, but that the increases were seen with one but not the other allele of each of the polymorphisms. These findings were plausible and linked to clinical status, as, for both CD14 and CC16, the allele for which there was no increase in plasma level was associated with more severe acute asthma. Because these findings could not be made studying individuals with stable asthma, they make an important contribution to current knowledge of the mechanisms of asthma. The two genes we studied were specifically selected because of their involvement in inflammatory pathways, but studies on similar cohorts should be extended to include a larger panel of genes, examining both individual SNPs and haplotype patterns.
As previously noted, CD14 is known to play an important role in the immune system and plasma levels were increased with acute asthma. However, the precise physiologic role of sCD14 is still unclear, as it may be beneficial as a scavenger to neutralize circulating LPS, or its absence on endothelial cells that do not express membrane-bound CD14 may allow LPS to produce an aggressive and harmful proinflammatory cytokine response. Increased serum levels of sCD14 have been reported in severe acute systemic conditions, such as gram-negative septicemia, polytrauma, burns, and acute respiratory distress syndrome and have been associated with increased morbidity and mortality (27–29). Studies of IC14, a CD14-specific monoclonal antibody, showed the potential of this treatment to limit the excessive systemic inflammatory response in subjects with severe sepsis (30). Consistent with these reports, the current study found that during acute asthma attacks in children, plasma sCD14 levels were inversely correlated with severity, suggesting a protective role for sCD14. Plasma sCD14 levels have been directly correlated with IFN- and inversely correlated with IL-4 (11), suggesting that in children with acute asthma, the beneficial effect of greater CD14 activity may result from increased Th1 and decreased Th2 responses. A relative predominance of Th1 over Th2 cytokines assists in the elimination of viral infections (31), thus lower levels of sCD14 may allow ongoing viral replication and inflammation.
Acute asthma attacks are associated with a marked increase in airway inflammation and this was reflected by the finding of mean CC16 plasma levels being 95.9% higher during the acute attacks compared with convalescence. Plasma levels of CC16 are reported to reflect CC16 levels in bronchoalveolar lavage in healthy adults (16). This study is the first to demonstrate altered circulating CC16 levels in subjects during an acute attack. The only other study comparing acute and convalescent plasma CC16 levels did not find a difference, but compared paired plasma CC16 levels in only 10 adults with asthma (18).
This study demonstrated that genotype-specific differences in plasma levels of sCD14 and CC16 during acute exacerbation were even more pronounced than during convalescence. For CD14, subjects with –159TT and –159CT genotypes had 42 and 31% higher plasma sCD14 levels, respectively, compared with 10% lower levels in those with –159CC. The finding that during acute asthma –159TT subjects had the highest sCD14 levels, heterozygotes intermediate, and –159CC the lowest levels was consistent in direction with those from a normal population of American school children (11). However, in contrast to the study by Baldini and colleagues, which suggested a dominant model, we found a linear effect, with each additional T allele having an additive effect on plasma sCD14 levels. Interestingly, this linear effect was only present during the acute asthma exacerbation, suggesting that gene expression at this time is likely to be exaggerated. Levels of sCD14 were similar for the different genotypes in convalescence, further demonstrating that this pathogenetic process could only have been identified by studying children during acute attacks. Our findings were also consistent with functional studies that showed that the T allele produced 32% greater activity than the C allele (7).
For CC16, subjects with 38GG and 38AG genotypes had 134% and 80% higher plasma CC16 levels, respectively, compared with 38AA subjects, who paradoxically had plasma CC16 levels that were 33% lower. These genotype-specific differences in plasma levels were also consistent in direction with the findings from a case control study of Australian children (25). However, in addition to having the lowest levels of CC16 during an acute asthma attack, subjects with 38AA appeared to have had a decrease in plasma CC16 levels when the degree of airway inflammation was maximal. This difference may reflect decreased gene expression by the A allele or that the consumption of CC16 greatly exceeds production during acute asthma. Subjects with asthma with the 38A allele may be unable to increase levels of CC16 in the airway at the time of viral infections when protection from excessive airway inflammation becomes most critical. This may result in 38AA subjects experiencing more severe acute asthma due to lower levels of this endogenous antiinflammatory agent.
Those subjects who were either CD14 –159CC or CC16 38AA were over three times more likely to have moderate or severe attacks of acute asthma compared with the other genotypes. A potential mechanism in determining severity of an acute attack is impaired ability to increase sCD14 and CC16. The effect of CD14 C-159T on asthma severity was partly dependent on plasma sCD14 levels, with higher levels associated with a milder asthma attack, possibly because of increased Th1 cytokine release. Although differences for CC16 did not reach statistical significance, mildly affected subjects tended to have the highest CC16 levels and severely affected the lowest. However, as CC16 levels in plasma are approximately 10,000 times lower than in bronchoalveolar lavage (32), any changes seen in the plasma are likely to be substantially more pronounced in the airway.
Although this study population was relatively large compared with other studies of children with acute asthma, the number of subjects with some genotypes, such as CC16 38AA, was relatively small compared with other genetic association studies. However, this study differed from previous studies in that the stimulation of inflammatory pathways was likely to have been near maximal and, for the genes assessed, the genes' own product was an important primary outcome variable. Also, genotype-specific plasma levels of sCD14 and CC16 were consistent with previous studies, as well as being internally consistent between the genotypes, and these consistencies strongly support the validity of the findings. Thus, the striking and consistent associations found, despite the relatively small numbers of subjects, vindicate this novel approach of studying genetic associations at a time when the loss of control of inflammation could be expected to be maximal. Such an approach has the potential to allow the identification of important genetic associations in relatively small cohorts.
Other potential limitations should be considered. Although children were studied early in the course of the acute attack, in all cases blood samples were obtained after the first dose of prednisolone. Exogenous steroids can promote down-regulation of sCD14 and up-regulation of CC16 (33, 34), but there is no evidence to suggest that steroids would have an allele-specific effect on the polymorphisms studied. Furthermore, the highly significant findings of genotype-specific variation in acute plasma levels of sCD14 and CC16 suggested that, even if steroids influenced the absolute level of gene product, genotype differences still play a major role in determining altered levels of gene product between individuals and asthma severity. Ethnicity is known to influence the genotype frequency for both CD14 C-159T and CC16 A38G (35). However, as more than 90% of our population were white Australians, of families originally from Europe, the low frequency of other ethnic origins meant that we were unable to investigate the effect of ethnicity in this study. In studying genetic associations, consideration must be also given to the presence of multiple SNPs per gene and to functional haplotypes. In the present study, only one SNP was examined for each gene, but CD14 C-159T and CC16 A38G are considered the most important CD14 and CC16 SNPs, as they are the only ones shown to have functional significance.
In summary, this study of children during acute asthma attacks identified significant relationships between two common promoter SNPs in the CD14 and CC16 genes and genotype-specific differences in their gene products' plasma levels, which in turn reflected the severity of the acute asthma attacks. These results suggest that in acute asthma production of both CD14 and CC16 is increased in an attempt to control airway inflammation, and for subjects whose genotype limits or prevents these increases, the ability to control airway inflammation is impaired, resulting in more severe asthma. Studies of this nature have the potential to provide new insight into mechanisms that control airway inflammation, and to facilitate the development of individually tailored treatments.
Acknowledgments
The authors thank the children and families who participated in this study and acknowledge the technical assistance of Jenny Tizard.
FOOTNOTES
Supported by grants from the National Health and Medical Research Council of Australia, Asthma Foundation of Western Australia, and the West Australian Institute of Medical Research.
Dr. Shahir Taheri is deceased.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200509-1367OC on December 30, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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