Abstract
CD14, a co-receptor for endotoxin, plays a significant role in the inflammatory response to this environmentally important pollutant. The C-159T single nucleotide polymorphism (SNP) in the CD14 gene promoter is reported to affect expression of CD14, with TT homozygous persons having higher CD14 expression. This SNP has been linked to pathogenesis of asthma and with cardiovascular diseases in smokers. We hypothesize that CD14 also plays a role in development of COPD in smokers who are exposed to inhaled endotoxin by cigarette smoking and to endotoxin released from Gram-negative microbes colonizing their airways. To assess the effect of the C-159T SNP of the CD14 gene promoter on lung function and GOLD score in smokers with COPD, we recruited 246 smokers with COPD with a range of 10–156 pack-year smoking exposures. We found that the C-159T single gene polymorphism of the CD14 gene promoter may play a role in modulating severity of obstructive impairment in smokers with COPD: The TT genotype was associated with lower lung function in smokers with a moderate smoking history. However, the CC genotype was associated with decreased lung function in heavy smokers (>56 pack-years). The result on CC genotype in risk for COPD is analogous with the effect of this genotype in risk for asthma. CD14 may be a factor in the pathophysiology of COPD, as it is in asthma and smoking-related cardiovascular diseases.
Introduction
Exposure to environmental endotoxin is an important factor in the development of a variety of airway diseases, including asthma and occupational lung disease. Response to low doses of inhaled endotoxin is mediated to a great extent by airway monocytes and macrophages. Endotoxin binds with CD14, a glycosylphosphatidylinositol-linked plasma-membrane glycoprotein which is abundantly expressed on monocytic cells and facilitates endotoxin ligation of TLR4 with ultimate activation of NF-κB1;2. CD14 expression on airway monocytes and macrophages is an important determinant of airway responses to inhaled endotoxin, as demonstrated in our report that the degree of neutrophil influx to the airway after endotoxin challenge correlated with CD14 expression on airway macrophages prior to challenge3.
Though the role of endotoxin exposure and CD14 has been extensively studied in other airways diseases, it does not appear to have been examined in COPD or chronic bronchitis. Endotoxin is a significant constituent of tobacco smoke4, and ambient air endotoxin is markedly increased in environments which include tobacco smoke5. Additionally, the airways of many smokers are infected or colonized with gram-negative bacteria, providing an endogenous source of endotoxin exposure to airway macrophages and monocytes6. Given the exposure of smokers to endotoxin in the airway, it seems likely that CD14 modulates chronic bronchitis and COPD.
In asthma7, occupational lung disease8, and post –lung transplant9, the commonly occurring C-159T polymorphism (also referred to as C-260T) of the CD14 gene promoter has been shown to be an important genetic modifier of airway disease. The T allele of this SNP is associated with increased production of CD14, and persons with the TT genotype would be expected to be more responsive to endotoxin. We have previously shown a CD14-modulated airway neutrophil response to low doses of inhaled LPS3. As compared to the CC and CT genotypes, the TT genotype for the CD14 gene promoter has been associated with increased serum levels of soluble CD1410 and increased response of PBMC from asthmatic children to endotoxin in vitro11. The T allele is linked to increased occurrence of lung disease in farmers8, to the protective effect of endotoxin exposure in decreasing risk for developing allergy and asthma, and to the risk of acute exacerbation of airway disease associated with high levels of environmental endotoxin1; 12–14.
Tobacco smoke exposure also impacts gene/environment interactions in asthma. Genome wide scans have shown that in children passively exposed to tobacco smoke, there is linkage between asthma and bronchial hyper- responsiveness and chromosome 5q, the location of the CD14 gene15. In Latino populations, CD14 SNPs were linked with asthma severity and IgE production in persons exposed to tobacco smoke16. Women also demonstrated an association between the CD14 C-260T genotype and total IgE levels that was modified by the level of endotoxin exposure, such that compared with CC and CT genotypes, the TT genotype was associated with lower serum IgE in individuals with very low environmental endotoxin exposure, but with higher serum IgE in individuals with higher endotoxin exposure17. Tobacco smoke exposure has also been shown to increase serum levels of soluble CD14 in children18. Though there are conflicting reports of the role of CD14 in cardiovascular disease (see review by Arroyo-Espliguoro et al. 2004)19, CD14 has been implicated as a risk factor for myocardial infarction, stroke, and cardiovascular diseases with greater risk being reported in smokers carrying the T allele20–22. Taken together, these observations suggest that CD14 may be an important biological factor in mediating the pathophysiology of diseases linked to tobacco smoke exposure.
We hypothesized that CD14 is a determinant of COPD disease severity regarding airway obstruction and symptoms associated with tobacco smoking. To explore this hypothesis, we assessed the role of the C-159T SNP of the CD14 gene promoter on lung function in 246 smokers identified as having COPD who were at least 40 years of age with at least a 10 pack-year history of tobacco use. As the TT genotype is associated with increased expression of CD14, our a priori hypothesis was that COPD patients who were homozygous for the T allele at the C-159T polymorphism would have decreased lung function relative to those with CT or CC genotypes. To our knowledge, this is the first report examining the effect of the CD14 gene promoter on lung function of smokers with COPD.
Methods
Subjects
Volunteers were identified via response to advertisements and recruitment from clinical research sites in Chapel Hill and Raleigh, NC. Written consent was obtained. To be included in this study, volunteers needed to be >40 years of age, have a smoking history of at least 10 pack-years, meet GOLD criteria for GOLD stages 0–3, and be willing to have spirometry performed. Volunteers were excluded if they had other diseases that would likely affect lung function (e.g., active asthma, autoimmune diseases, immunodeficiencies). Volunteers consented to collection of DNA for genotyping using buccal swab techniques or blood collection of lymphocytes if significant oral pathology was observed. This study was reviewed and approved by the UNC Committee for the Protection of the Rights of Human Subjects (IRB).
Study Endpoints
Spirometry measures included the forced vital capacity (FVC), the forced expiratory volume at one second (FEV1), and the FEV1/FVC ratio. The FEV1 and FVC were expressed as the % of predicted normal values developed by Knudson et al, accounting for gender, ethnicity, height, weight and age23. GOLD (Global Initiative for Obstructive Lung Disease) staging was done, with spirometric parameters being determined after use of inhaled albuterol, except in GOLD stage 0 subjects: Stage 0: At Risk: chronic cough and sputum production but normal spirometry. Stage 1: Mild COPD: mild airflow limitation (FEV1/FVC < 70% but FEV1 > 80% predicted) and usually, but not always, chronic cough and sputum production. Stage 2: Moderate COPD: worsening airflow limitation (FEV1/FVC <70% but 30% < FEV1 < 80% predicted) and usually the progression of symptoms, with exertional shortness of breath. Stage 3: Severe COPD: severe airflow limitation (FEV1 < 30% predicted) or the presence of respiratory failure or clinical signs of right heart failure. Genotyping for the C-159T polymorphism of the CD14 gene promoter was performed as previously described3; 10.
Statistical Analysis
The primary hypothesis of this study was that smokers with COPD with the TT genotype for the C-159T single nucleotide polymorphism of the CD14 gene promoter would have decreased lung function and increased GOLD scores relative to those with the CT and CC genotypes. To explore the potential effect of gene/environment interaction of smoking in development of COPD, we conducted a formal statistical investigation of the potential interaction using multiple-regression techniques24. In particular, a linear regression model was fitted for quantitative endpoints FEV1 (%predicted), FVC (%predicted), FEV1/FVC ratio, and a logistic regression model was fitted for the GOLD scores (0 for GOLD 0 or 1, and 1 for GOLD 2 or 3). All analyses were conducted with adjustment for subject’s current smoking status, Age, Gender (Male=1, Female=0), BMI and Race. Because there are three genotypes to consider (TT, CT, CC), we used two dummy variables to model the CD14 genotype (CC as the reference group). We centered the pack-year history variable in all regression analyses.
We built a sequence of nested models starting with a full model that includes nonlinear terms of smoking history (using the spline technique), genotype, interaction terms between these two factors, and the adjustment factors noted above. We then sequentially reduced the full model using the maximum likelihood ratio test as model selection criteria to arrive at the final model. The final models with quadratic terms of pack-year history are not statistically different from models using the spline fitting. All analyses were carried out using the SAS version 9.1 (SAS Institute Inc., Cary, NC)25. Analyses to test for interaction between genotype and pack-year history were also carried out for distinct sub-groups which represented a sizable fraction of the total study cohort, including current-smokers, ex-smokers and Caucasians.
Results
General Findings
246 volunteers were recruited, with genotype, spirometric and clinical information obtained from these volunteers. The demographic characteristics of the entire volunteer group, as well as COPD characterization stratified on the basis of genotype for the C-159T SNP for the CD14 gene promoter are shown in Table 1 and 2. Although there were no statistically significant differences observed between the means of the COPD outcomes (FEV1 %predicted, FVC %predicted, FEV1/FVC ratio) across the three genotype groups, the TT group appears to have a lower mean FEV1 (% predicted), lower proportion of males, and lower mean FVC (% predicted), coupled with a higher proportion of Caucasians and a lower mean pack-year history, than the CC and CT groups, suggesting the need to account for these factors in the analysis and that an interaction effect may exist. Not unexpectedly, there were strong correlations between pack year history and the % of predicted FEV1 (r= −0.43, p<0.0001), % of predicted FVC (r= −0.38, p<0.0001), the FEV1/FVC ratio (r= −0.35, p<0.0001) and GOLD score (r= 0.38, p<0.0001) within the entire cohort. When segregated on the basis of genotype, these correlations for the CC and CT genotypes were similar to those for the overall group, whereas those for the TT genotype were not statistically significant.
TABLE 1.
Demographics and characteristics of study participants stratified by the CD14 C-159T genotype information
| All individuals (n = 246) |
genotype CC (n = 78) |
genotype CT (n =118) |
genotype TT (n = 50) |
|
|---|---|---|---|---|
| CD14 C-159T genotype (%) | - | 31.7 (78/246) | 48.0 (118/246) | 20.3 (50/246) |
| Age, mean ± SD | 53.9 ± 8.9 | 54.6 ± 9.1 | 53.1 ± 8.7 | 54.8 ± 9.0 |
| Male Gender (%) | 54.1 (133/246) | 64.1 (50/78) | 49.6 (58/118) | 50.0 (25/50) |
| Caucasian (%) | 83.7 (206/246) | 79.5 (62/78) | 82.2 (97/118) | 93.9 (46/49) |
| BMI, mean ± SD | 28.2 ± 5.8 | 27.3 ± 5.0 | 28.6 ± 6.3 | 28.6 ± 5.8 |
| Pack Year, mean ± SD | 45.1± 26.3 | 46.8 ± 29.7 | 45.8 ± 25.7 | 41.2 ± 21.5 |
| Current smokers (%) | 65.0 (160/246) | 30.8 (49/160) | 50.3 (80/160) | 18.9 (30/160) |
| Former Smokers (%) | 35.0 (85/246) | 34.1 (29/85) | 43.5 (37/85) | 22.4 (19/85) |
| FEV1 (%predicted), mean ± SD | 76.0 ± 27.5 | 76.1 ± 29.0 | 76.7 ± 27.8 | 74.0 ± 24.6 |
| FVC (%predicted), mean ± SD | 92.1 ± 21.4 | 91.7 ± 22.6 | 94.3 ± 20.7 | 87.3 ± 21.0 |
| FEV1/ FVC ratio, mean ± SD | 0.66 ± 0.16 | 0.66 ± 0.17 | 0.65± 0.16 | 0.66 ± 0.15 |
| Gold Score (%) | ||||
| 1 | 11.8 (29/246) | 31.0 (9/29) | 62.1 (18/29) | 6.9 (2/29) |
| 2 | 15.0 (37/246) | 16.2 (6/37) | 54.1 (20/37) | 29.7 (11/37) |
| 3 | 14.2 (35/246) | 37.1 (13/35) | 40.0 (14/35) | 22.9 (8/35) |
| 4 | 6.5 (16/246) | 43.8 (7/16) | 43.8 (7/16) | 12.5 (2/16) |
TABLE 2.
Fitted regression model for the association between pack-yr, genotypes and the FEV1/FVC ratio (Full data adjusted analysis)
| Parameter | Estimate | Standard Error | p-value |
|---|---|---|---|
| Intercept | 0.7541 | 0.0790 | <.0001 |
| TT | 0.0019 | 0.0245 | 0.938 |
| CT | −0.0235 | 0.0196 | 0.231 |
| Pack_yr | −0.0034 | 0.0007 | <.0001 |
| Pack_yr_sq | 0.00002 | 0.00001 | 0.013 |
| Pack_yr * TT | 0.0022 | 0.0010 | 0.030 |
| Pack_yr * CT | 0.0016 | 0.0007 | 0.022 |
| Current_smokers | 0.0417 | 0.0183 | 0.024 |
| Age | −0.0043 | 0.0011 | <.0001 |
| Gender | −0.0316 | 0.0176 | 0.073 |
| Caucasian | −0.0706 | 0.0238 | 0.003 |
| BMI | 0.0064 | 0.0015 | <.0001 |
Stratified by genotypes, the distributions for FEV1, FVC, FEV1/FVC per pack-year quartile are depicted in Figure 1. In the first quartile, there was no difference in any of the spirometric endpoints between the three genotype groups. In the second quartile, the TT and CT groups had decreased lung function while the CC group held steady. In the third quartile the TT group continued to decrease while the CC group increased toward the CT group. Finally, in the upper pack-year quartile, the CC genotype group exhibited the greatest decrease in lung function while TT group held steady. Figure 2 presents a simple non-parametric smoothing fit, stratified by genotypes, for relationship between the pack-year histories in continuous scale to the levels of FEV1, FVC and FEV1/FVC, respectively. The nonlinear trend observed in Figure 1 is now much clearer with a continuous scale of the pack-year history. This again suggested a potential interaction effect between the pack-year and CD14. Considering that the pack-year history ranged from 10–153, we hypothesized that there may be a gene by environment (CD14 gene promoter vs. pack-year history) effect on lung function in this cohort of COPD patients. To investigate this possibility, we employed a multiple-regression model to formally test the potential interaction.
Figure 1. Box plots across four quartiles of pack-year history, by genotype, for FEV1 (%predicted), FVC (%predicted), and FEV1/FVC ratio levels.
Quartiles of the pack-year history are defined as 10–28 pack yr smoking for 1st quartile (Q1); 28–38 pack yr for Q2; 38–56 pack yr for Q3 and 56–153 pack yr for Q4. The horizontal line in the interior of the box is located at the median of the data. The height of the box is equal to the interquartile distance (IQD) which is the difference between the third quartile of the data and the first quartile. The IQD indicates the spread or width of the distribution for the data. The whiskers extend to the extreme values of the data or a distance 1.5*IQD from the center, whichever is less. Data points which fall outside the whiskers may be outliers.
Figure 2. Non-linear smoother fit for FEV1 (%predicted), FVC, and FEV1/FVC ratio by genotype (Full data set).
The lines in the figures are the running means depicting the nonlinear relationship between the pack-year and COPD endpoints across different genotypes. The FEV1/FVC ratio is tested to be significant in the full data set while both FEV1 and FEV1/FVC ratio are tested significant in the ex-smoker subpopulation.
Full Data Set Analysis
Interaction of Tobacco Exposure and Genotype on % Predicted FEV1
To determine if there is any interaction between pack-year history and genotype on % predicted FEV1, we built a sequence of nested regression models that include the interaction effects and used the maximum likelihood ratio test for testing. Although we confirmed there is a nonlinear relationship between % predicted FEV1 and pack-year history, we did not find a statistically significant interaction for the CD14 genotype and smoking.
Interaction of Tobacco Exposure and Genotype on % of predicted FVC
The same modeling strategy for assessing the interaction between pack year history and genotype on % of predicted FEV1 and FEV1/FVC ratio was employed. Again pack-year history is negatively associated with % of predicted FVC (β = −0.223, p < 0.0001), but we did not find effects of CD14 genotype on the relationship between pack-year history and FVC.
Interaction of Tobacco Exposure and Genotype on FEV1/FVC Ratio
To assess the interaction between pack-year history and genotype on the FEV1/FVC ratio, we employed the same statistical strategy described for FEV1 to arrive at the final model for FEV1/FVC ratio. The final model included a nonlinear pack-year term. We observed a significant interaction between the CD14 genotype and the effect of smoking history on FEV1/FVC ratio. The p-value for testing the interactions between pack-year genotype, based on 2 degrees of freedom (for testing TT and CT vs. CC), is p=0.03. CC genotype individuals have significantly and progressively decreasing FEV1/FVC ratio with increasing pack-year history, whereas the CT and TT genotypes appeared to reduce the impact of pack-years of smoking on lung function. Other significant factors are subject’s age (β = −0.0043, p<.0001), Male gender (β = −0.032, p=0.073), Caucasian race (β = −0.071, p=0.003), current smoking status (β = 0.042, p<.024), and BMI (β = 0.0064, p<.0001). These findings are shown in Table 3.
TABLE 3.
Fitted regression model for the association between pack-yr, CD14 genotypes and lung function (Ex-smoker adjusted Analysis)
| Predictor Variables‡ |
Fitted model for FEV1 (% predicted) |
Fitted model for FEV1/FVC Ratio |
||||
|---|---|---|---|---|---|---|
| Parameter Estimate |
Standard Error |
P value | Parameter Estimate |
Standard Error |
P value | |
| Intercept | 101.168 | 23.586 | <.0001 | 0.615 | 0.144 | <.0001 |
| Pack Year | −0.813 | 0.173 | <.0001 | −0.005 | 0.001 | <.0001 |
| CD14 C-159T genotype TT | −6.028 | 6.176 | 0.332 | −0.015 | 0.038 | 0.701 |
| CD14 C-159T genotype CT | 0.016 | 5.252 | 0.998 | −0.029 | 0.032 | 0.368 |
| Pack Yr ×TT | 0.450 | 0.231 | 0.055 | 0.003 | 0.001 | 0.034 |
| Pack Yr ×CT | −0.113 | 0.180 | 0.531 | 0.0003 | 0.001 | 0.762 |
| Pack Yr2 | 0.009 | 0.003 | 0.001 | 4.2E-5 | 1.5E-5 | 0.007 |
| Age | −0.689 | 0.300 | 0.025 | −0.004 | 0.002 | 0.039 |
| Male | −5.867 | 4.891 | 0.234 | −0.062 | 0.030 | 0.043 |
| Caucasian | −9.206 | 7.769 | 0.240 | −0.091 | 0.048 | 0.060 |
| BMI | 0.552 | 0.509 | 0.282 | 0.011 | 0.003 | 0.0004 |
The FEV1/FVC ratio is 0.042 units higher in current smokers than in ex-smokers. This might reflect the higher age of the ex-smokers as well as the possible increased motivation to stop smoking among more impaired smokers. As expected, older individuals have a reduced FEV1/FVC ratio (0.043 units per 10 years). BMI and gender appear to be significantly related to the FEV1/FVC ratio such that higher BMI is associated with increased FEV1/FVC ratio (0.064 units for a 10 units increase in BMI) while men have a lower FEV1/FVC ratio (0.032 units) as compared with women.
Interaction of Tobacco Exposure and Genotype on GOLD score
To model the effect of tobacco exposure and genotype on GOLD score, GOLD stages of 0 or 1 were assigned a value of 0 and GOLD stages of 2 or 3 were assigned a value of 1. Logistic regression analysis was then employed to evaluate the effect of these factors and their interactions on the GOLD outcome. A significant effect of pack-year history on GOLD score was observed (β = −0.0029 p < 0.0001), but genotype did not significantly modify this effect.
Caucasian (n=206) subpopulation analysis
Recognizing that the majority of the study population is Caucasian (83.7%) and that the Caucasian proportion in the three genotypes is not the same (Table 1), the interaction effects observed in the full data set may in some way reflect a possible interaction between race and smoking effect, we repeated the above analyses for the Caucasian subpopulation. These analyses yielded almost the same results as the full data set for FEV1% predicted, FEV1/FVC ratio and GOLD score.
Ex-smoker (n=85) subpopulation analysis
The same statistical strategy was employed in analysis of the ex-smoker subpopulation (~35% of total population). In this subgroup, we found a marginally significant interaction between the pack-year history and genotype on % predicted FEV1 as well as on FEV1/FVC ratio. The basic findings for FEV1/FVC ratio in the ex-smokers are similar to that of the full data set. The p-value for testing the interaction between the pack-year and genotype (TT vs. CC) was 0.03 while the p-value for testing the interaction for TT and CT vs. CC was 0.055 for the FEV1/FVC ratio. The final fitted models were given in Table 4. For the FEV1 % predicted, the ex-smoker population exhibits a much stronger, borderline statistically significant interaction between the genotype and pack year history (p=0.054 for testing TT and CT vs. CC). The final fitted models were given in Table 4. There is no significant interaction for the GOLD score analysis.
TABLE 4.
Ex-smoker Analysis: Final fitted models for %predicted FEV1 and FEV1/FVC ratio
| Predictor Variables‡ |
Fitted model for FEV1 (% predicted) |
Fitted model for FEV1/FVC Ratio |
||||
|---|---|---|---|---|---|---|
| Parameter Estimate |
Standard Error |
P value | Parameter Estimate |
Standard Error |
P value | |
| Intercept | 101.168 | 23.586 | <.0001 | 0.615 | 0.144 | <.0001 |
| Pack Year | −0.813 | 0.173 | <.0001 | −0.005 | 0.001 | <.0001 |
| CD14 C-159T genotype TT | −6.028 | 6.176 | 0.332 | −0.015 | 0.038 | 0.701 |
| CD14 C-159T genotype CT | 0.016 | 5.252 | 0.998 | −0.029 | 0.032 | 0.368 |
| Pack Yr ×TT | 0.450 | 0.231 | 0.055 | 0.003 | 0.001 | 0.034 |
| Pack Yr ×CT | −0.113 | 0.180 | 0.531 | 0.0003 | 0.001 | 0.762 |
| Pack Yr2 | 0.009 | 0.003 | 0.001 | 4.2E-5 | 1.5E-5 | 0.007 |
| Age | −0.689 | 0.300 | 0.025 | −0.004 | 0.002 | 0.039 |
| Male | −5.867 | 4.891 | 0.234 | −0.062 | 0.030 | 0.043 |
| Caucasian | −9.206 | 7.769 | 0.240 | −0.091 | 0.048 | 0.060 |
| BMI | 0.552 | 0.509 | 0.282 | 0.011 | 0.003 | 0.0004 |
Current-smoker (n=160) subpopulation analysis
There is still a nonlinear relationship between both the % predicted FEV1 and the FEV1/FVC ratio, however, our analysis based on the current-smoker subpopulation reveals no interaction between genotypes and pack year history for the FEV1/FVC ratio and the % predicted FEV1. There are also no significant interactions for the GOLD score analysis.
Discussion
In this study, we tested the hypothesis that smokers with chronic bronchitis or COPD who also had the TT genotype for the C-159T nucleotide polymorphism for the CD14 gene promoter would have greater obstructive impairment of lung function (FEV1, FEV1/FVC ratio) and increased disease severity (GOLD score) relative to those with the CC or CT genotypes. This hypothesis was developed on the basis of observations in asthma14, occupational lung disease8 and studies of acute response to endotoxin challenges in normal volunteers and asthmatics3; 26. In farmers occupationally exposed to endotoxin-containing bioaerosols, the TT genotype was associated with decreased lung function, including %predicted FEV18. Regarding endotoxin, we have reported that the acute airways inflammatory response to inhaled low-dose endotoxin is correlated positively with the pre-challenge or constitutive level of expression of mCD14 on airway macrophages3.
When stratifying the population only on the basis of genotype, without accounting for smoking history or other demographic features (such as gender, ethnicity, age), we observed no difference in lung function of smokers with COPD on the basis of genotype. However, given the gene by environment interactions described for development of allergic airway disease for the C-159T nucleotide polymorphism for the CD14 gene promoter relative to environmental endotoxin exposure and the increased exposure of smokers to inhaled endotoxins, it seemed plausible that genetically based differences in lung function due to smoking might emerge when the environmental exposure (pack-year history) was considered.
To more completely describe and investigate the relationship between pack-year history and lung function in persons with different genotypes for the C-159T SNP for the CD14 gene promoter, we employed regression modeling approaches which would take into account the demographic features across a continuous pack-year smoking history measure. These analyses indicate that at lower pack-year smoking histories the TT genotype is associated with increased risk for decreased lung function (FEV1/FVC ratio). However, among smokers with higher pack-year histories, the CC genotype confers greater risk. Overall, these data suggest that there is a complex gene-by-environment interaction of the CD14 gene promoter on lung function in smokers with COPD.
While our results are generally consistent with our hypothesis in that persons with the TT genotype appear initially more susceptible to the effect of smoking on lung function, the CC genotype seems to have a deleterious effect at higher pack-year exposures. However, smoking itself has been reported to modulate expression of CD14 on airway macrophages irrespective of CD14 genotypes. Smoking may also modify the effect of a given genotype on production of soluble CD14. In a study examining serum levels of soluble CD14 (sCD14), the T allele was associated with increased levels of sCD14, though this was not observed in smokers, suggesting that the effect of the T allele on CD14 production is muted in smokers22. In asthma, smoking also influences the effect of the CD14 gene promoter in disease and development of IgE responses, and airway hyperreactivity and asthma has been linked to the chromosome 5q region (which contains the CD14 gene)15. Also, the effect of the T allele of the CD14 gene promoter on likelihood for developing antigen–specific IgE responses was more pronounced in smokers27. Thus, while the effect of smoking on CD14 expression and the genetic regulation of such expression is incompletely understood, it seems likely that smoking modifies the effect of the C-159T SNP on CD14 expression.
As noted previously, at higher pack-year exposures the CC genotype appears to confer increased risk for impairment of lung function relative to the TT genotype. This would be consistent with the effect of the CC genotype on risk for disease in asthma, as shown by genotype-specific increases in both development of antigen-specific IgE responses and wheeze in non-atopic individuals12; 16. The CC genotype is also a risk factor for smoking-related non-respiratory diseases. In examining the effect of smoking on carotid atherosclerosis, those with the CC genotype had increased risk of diseased carotids. In that study, the authors speculated that this was due to decreased levels of sCD14 in the serum of these patients28; 29. As with the studies of CD14 genotype effects on carotid artery disease, our study is limited because we did not assess either serum or airway levels of sCD14, or CD14 expression on airway or circulating monocytes or macrophages from our volunteers. Thus we did not examine the CD14 expression phenotype relative to the C-159T SNP for the CD14 gene promoter in these smokers.
To our knowledge, this is the first examination of the effect of the C-159T SNP of the CD14 gene promoter on progression of COPD in smokers with the chronic bronchitis form of COPD. Our findings suggest that there is a complex gene-by-environment interaction for the effect of the CD14 gene promoter on smoking-induced changes in lung function. The TT genotype appears to be associated with increased risk for obstructive impairment with moderate degrees of smoking (29–57 pack years), whereas the CC genotype is associated with increased risk for loss of lung function with heavy smoking (>57 pack years). While the molecular mechanisms which mediate this effect are unknown, our results suggest that innate immune mechanisms may play an significant role in disease modulation in smoking- induced airway obstruction in persons with COPD. These data are also consistent with the idea that tobacco smoke exposure may be able to modify a number of gene/environment interactions in airway diseases. These results provide a strong rationale for including the C-159T SNP of the CD14 gene promoter among other candidate polymorphisms when studying the genetics of lung disease related to smoking or to inhalation of endotoxin.
Acknowledgments
Funded by: NIH RO1-HL66559, P50-HL084934
We thank Dr. William Reed for generating the primers and Mr. Fernando DeMeo for performing the genotyping.
Footnotes
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Conflict of Interest Statement
None of the authors have a conflict of interest to declare in relation to this work.
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