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Published ahead of print on July 24, 2006, doi:10.1164/rccm.200603-443OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 875-885, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200603-443OC

Original Article

A Single Nucleotide Polymorphism in the CCL1 Gene Predicts Acute Exacerbations in Chronic Obstructive Pulmonary Disease

Noriaki Takabatake, Yoko Shibata, Shuichi Abe, Toshihiro Wada, Jun-ichi Machiya, Akira Igarashi, Yoshikane Tokairin, Guijin Ji, Hidenori Sato, Makoto Sata, Yasuchika Takeishi, Mitsuru Emi, Masaaki Muramatsu and Isao Kubota

First Department of Internal Medicine, Yamagata University School of Medicine, Yamagata; HuBit Genomix, Inc., Tokyo; and Medical Research Institute, Tokyo Medical & Dental University, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Noriaki Takabatake, M.D., First Department of Internal Medicine, Yamagata University School of Medicine. 2-2-2, Iida-Nishi, Yamagata 990-9585, Japan. E-mail: takabata{at}med.id.yamagata-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Acute exacerbations (AEs) in chronic obstructive pulmonary disease (COPD) are a major cause of morbidity and mortality in COPD.

Objectives: The marked heterogeneity in the host defense mechanisms may be attributed to single nucleotide polymorphisms (SNPs) in the inflammatory chemokines that show enhanced expression in the airway of patients with COPD who experience AEs.

Methods: We investigated four SNPs of the CCL11, CCL1, and CCL5 genes in relation to the frequency and severity of AEs in retrospective and prospective studies of a cohort of 276 male patients with COPD.

Measurements and Main Results: In the 2-yr retrospective study , one SNP (National Center for Biotechnology Information SNP reference: rs2282691) in the predicted enhancer region of the CCL1 gene, encoding a chemotactic factor for a series of leukocytes, was significantly associated with the frequency of AEs in a dominant model (Fisher's exact test: odds ratio [OR], 2.70; 95% confidence interval [CI], 1.36–5.36; p = 0.004; logistic regression: OR, 3.06; 95% CI, 1.46–6.41; p = 0.003; and Kruskal-Wallis test: p = 0.003). In the 30-mo prospective study, the "A" allele was a significant risk allele for the severity of AEs, with a gene–dosage effect (Kaplan-Meier method with log-rank test: AA vs. TT; log-rank statistic: 7.67, p = 0.006; Cox proportional hazards regression method: OR, 5.93; 95% CI, 1.28–27.48; p = 0.023). The electromobility shift assay showed that C/EBPbeta, a key transcriptional factor in response to pulmonary infections, binds to the "T" allele, but not to the "A" allele.

Conclusions: Variants in the CCL1 gene are associated with susceptibility to AEs through their potential implication in the host defense mechanisms against AEs.

Key Words: acute exacerbations • C/EBP&beta • • chronic obstructive pulmonary disease • inflammatory chemokine • single nucleotide polymorphisms

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality throughout the world, and is projected to be the third most common cause of death and the fifth most important cause of chronic disability by 2020 (1, 2). Acute exacerbations (AEs) in COPD are a common cause of morbidity (3, 4) and mortality (5, 6) in patients with COPD, and place an enormous burden on health care resources. AEs are defined by acute worsening of symptoms from the usual stable state, increased airway inflammation, and physiologic deterioration (7–9). AEs in patients with COPD are now recognized to have serious consequences for both the individual and society. The negative impact of the disease in such patients, as assessed by health status measurements, is significantly greater in those with more frequent exacerbations than in those who rarely experience exacerbations. This holds true even when the exacerbation does not require hospital treatment (10). In addition, hospitalization due to AEs is the single greatest component of the overall costs of COPD.

Infections are the most common precipitant of AEs (11). AEs are heterogeneous disorders regarding their underlying mechanisms for their development, and host defense factors against pathogens, either bacterial or viral, have been suggested to play significant roles in recurrent AEs for the following reasons: First, both the cell-mediated and humoral immune responses against the offending pathogens vary among patients and may even become blunted in a substantial number of patients. This deficient cell-mediated immune response is associated with the occurrence of exacerbations (12, 13). Second, between 25 and 50% of patients with COPD have lower airway colonization by bacteria, implying a breach of host defense mechanisms. The presence of bacterial colonization in patients with COPD is likely to have an influence on the exacerbation frequency and on the character and severity of exacerbations (11). Third, although there is a relationship between exacerbation frequency and sputum inflammatory cytokine levels during a stable period, such as levels of interleukin (IL)-6 and IL-8 (7), there is considerable variability in inflammatory markers during an exacerbation, suggesting marked heterogeneity in the degree of the inflammatory response during an AE (7, 14, 15). Collectively, individual susceptibility factors to exacerbations, due, at least in part, to the genetic component, might contribute to the heterogeneous features of AEs in COPD. In fact, several longitudinal cohort studies have demonstrated a wide distribution of the frequency of AEs (16–18).

Chemokines are a small group of chemotactic cytokines with molecular weights in the range of 8 to 10 kD, and they have been subdivided into four subfamilies on the basis of structure (19, 20). Chemokines constitute a family of mediators of inflammation and immunity, and are specifically involved in the regulation of immune defense and in the housekeeping of the immune system (19, 20). Inflammatory chemokines are secretory proteins produced by many different cell types and by immigrating leukocytes in response to bacterial toxins and inflammatory cytokines, such as IL-1beta, tumor necrosis factor-{alpha}, and IFN-{gamma}, and exert their effects locally in a paracrine or autocrine fashion (19, 20). In addition to their leukocyte attracting function, inflammatory chemokines play a pivotal role in immunomodulation (up-regulating both T cells and antigen-presenting functions), potentiation of antigen-specific T-helper 1 (Th1) and Th2 clone activation, and promotion of cellular proliferation, including the release of various lymphokines (19, 20). Although several chemokines play important roles in the airway of patients who experience AEs (21–23), the relationship between the effects of these chemokines and the mechanisms underlying the heterogeneous features of these AEs in COPD—in particular, individual susceptibility factors due to potential genetic polymorphisms—remains unknown.

Pathologically, COPD is characterized by chronic inflammation and remodeling throughout the conducting airways, parenchyma, and pulmonary vasculature (24–27). In stable COPD, there is a characteristic infiltration of the bronchial mucosa with increased numbers of CD8+ T lymphocytes and monocytes/macrophages (28–31). However, in exacerbations in mild COPD, there is an accumulation of eosinophils in the mucosa, due to the up-regulation of eotaxin (CCL11) and RANTES (regulated on activation, normal T-cell expressed and secreted; CCL5), which are the two representative eosinophil chemoattractants and strongly expressed in both the surface epithelium and subepithelial lymphomononuclear cells (21, 22). This "allergic" profile of inflammation in exacerbations characteristic of mild COPD is replaced by an increase in the number of neutrophils in exacerbations in severe COPD. This is likely to be due to the up-regulation of neutrophil chemoattractants, such as IL-8 (CXCL8), and epithelial-derived neutrophil attractant-78 (CXCL5) (23). In addition, exacerbation frequency increases with disease severity (32).

Taking these backgrounds into account, we hypothesized that the individual susceptibility to AEs may be attributed to the genetic variance in inflammatory chemokines that show enhanced expression in the airway of patients who experience AEs in various stages of COPD. The genes of CCL and CXCL chemokines, the major two subfamilies, tend to be clustered on the same chromosome locus (17q11.2-q12 and 4q21.1, respectively). Thus, as an initial step, we sought to identify single nucleotide polymorphisms (SNPs) in genes of CCL11, CCL5, and CCL1 (a chemotactic factor for a series of leukocytes including monocytes/macrophages) (33–39), all of which are closely located on the same region in the human genome, and to determine their relation with regard to the frequency and severity of AEs in retrospective and prospective studies of a cohort of 276 male patients with COPD. We selected the CCL1 gene together with CCL11 and CCL5 genes because the CCL1 protein is not only a monocyte/macrophage chemoattractant (33) but also activates Th2 cells, such as Th2-polarized T lymphocytes and natural killer (NK) cells, and antigen-elicited eosinophils (34–39). The allergic profile of inflammation is one of the characteristic features in exacerbations of mild COPD (21, 22). In addition, the SNPs beside the CCL11 and CCL5 genes were required to conduct haplotype block analysis for disease association, if we could demonstrate local linkage disequilibrium (LD) (40–42). We found four SNPs in these genes by searching the nucleic acid database. Therefore, the purpose of this study was to identify SNPs of CCL11, CCL1 and CCL5 that may render patients susceptible to AEs, and thereby attempt to predict occurrence of AEs during the course of the disease.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Population
Japanese patients with COPD who had consulted the outpatient clinic of the University of Yamagata between 2001 and 2002 were recruited for the study. We enrolled 291 patients with COPD, and ultimately analyzed 276 male patients whose genomic DNA was available for genotyping. COPD was diagnosed according to the criteria established by the NHLBI/World Health Organization workshop summary (2). Irreversible chronic airflow obstruction was confirmed by spirogram for all patients. Fifty of 276 patients with COPD had history of atopy, such as attacks of shortness of breath with wheezing, or increased blood eosinophils. Patients with concomitant diseases, such as chronic heart failure, autoimmune disorders, or malignant diseases, were excluded. All of the patients were ex-smokers. None of the patients were receiving nutritional support therapy. All of the patients received influenza vaccination every winter. After an overnight fast, all subjects underwent anthropometric measurements. The study protocol was approved by the local ethics committee of Yamagata University School of Medicine, and written, informed consent was obtained from all subjects before participating in this study.

Pulmonary Function Test
Forced vital capacity and FEV1 were measured with standard spirometric techniques (CHESTAC-25 part II EX; Chest Corp., Tokyo, Japan). The highest value from at least three spirometric maneuvers was used. Reference values were those proposed by Quanjer (43). Arterial blood gas was analyzed with or without the subject breathing supplemental oxygen in the sitting position (280 Blood Gas System; Ciba Corning Diagnostics Corp., Medfield, MA).

Candidate SNP Selection and Polymorphism Genotyping
The characteristics of the genes and SNPs (SNP ID 1–4) used for the test are shown in Table 1. These SNPs were extracted as described previously (44). To identify putative transcriptional factor binding sites in sequences surrounding SNPs, the computer program Match (version 1.4.1), distributed with the Transfac Professional database (Biobase Biological Databases, Tokyo, Japan), was used, applying 85% as a target threshold score (Tables 2 and 3) (45–48). The chromosomal locations of CCL11, CCL1, and CCL5 are illustrated in Figure 1. Altogether, four SNPs derived from these three genes were tested for the association study. Analysis of genetic polymorphism SNP genotyping was performed by TaqMan allelic discrimination assay, with minor modifications (44, 49). Several data (signal intensity) were eliminated to preserve the reliability of the assay system (missing data were less than 5%), because recurrent low signals in genotyping rarely occur in this assay and such signals are not always reliable. The alleles and the genotype frequencies of the tested SNPs were determined and combined with the clinical data to conduct statistical analysis.


Figure 1
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  		Figure 1. Chromosome 17 (National Center for Biotechnology Information [NCBI] built 34). The chromosomal locations of the genes and single nucleotide polymorphisms (SNPs) of the inflammatory chemokines used for the study are illustrated. These genes (CCL11, CCL1, and CCL5) are clustered on the same chromosome locus (17q11.2-q12), and the four SNPs (SNP ID: 1–4; Table 1) were extracted as described previously (44).

 

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  		TABLE 1. FOUR POLYMORPHISMS IN CCL GENES EXAMINED IN THE STUDY

 

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  		TABLE 2. FOUR POLYMORPHISMS IN CCL GENES AND PUTATIVE TRANSCRIPTIONAL FACTOR BINDING SITES (A ALLELE)

 

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  		TABLE 3. FOUR POLYMORPHISMS IN CCL GENES AND PUTATIVE TRANSCRIPTIONAL FACTOR BINDING SITES (B ALLELE)

 
Retrospective and Prospective Cohort Studies
Before the prospective study, retrospective data were collected from patients with COPD in our institute at the University of Yamagata between January 1, 2001, and December 31, 2002. As of January 1, 2003, a total of 276 male patients with COPD were eligible for blood sampling and evaluation of the frequency of AEs according to their individual medical records. The definition of an AE is not yet firmly set (50); however, in the current retrospective study, we made the diagnosis of AEs as follows. Clinically, an episode was considered to represent an AE if the patient met the following three conditions: (1) change in sputum (color, texture, or quantity); (2) occurrence of at least one additional symptom among increased shortness of breath, cough, or presence of fever; and (3) evidence of the nontrivial nature of the episode (as determined by either an unscheduled hospital visit and/or use of antibiotics). In addition, to avoid bias from the patients and to increase objective evaluation for the physicians, the evidence of an AE must be confirmed by clear laboratory finding(s), such as increased C-reactive protein level or increased peripheral blood leukocyte count, and radiographic finding(s) such as pneumonia (51). According to these criteria, in the retrospective study, two independent pulmonary physicians carefully assessed the medical records of each individual during the past 2 yr, and only the cases that both physicians agreed were AEs in COPD were counted. In this retrospective study, four patients were ultimately excluded because of lack of precise information on the incidence of the AEs.

Overall, in this manner of retrospective study, 451 AEs were identified by at least one physician, and both physicians ultimately agreed on 388 cases of AEs. Therefore, the concordant rate for each exacerbation between the two physicians was 86.0%. Also, of 272 patients, disagreement over a total incidence of AEs was identified in 49 patients. Therefore, the concordant rate for each patient with respect to a total incidence of AEs was 82.0%.

In the prospective study, a total of 276 patients were followed up in the outpatient clinic every month and also at the time of suspected exacerbations. At each routine visit, a thorough clinical evaluation was performed by a general physician, and, for a patient suspected of having an exacerbation, one of the study physicians (pulmonary physicians) joined the general physician for the clinical evaluation. In the current prospective study, we made the diagnosis of an AE as follows. The examiner asked six questions about overall well-being, dyspnea, cough, sputum production, sputum viscosity, and sputum purulence. Emphasis was placed on grading three cardinal symptoms relative to the baseline: volume of sputum production, sputum purulence (color), and dyspnea. The patient then was evaluated with regard to overall appearance, respiratory rate, wheezes, and rales. On the basis of these observations, clinical determination was made on whether the patient was in clinically stable condition or was having an exacerbation (12, 13). In addition, the evidence of AEs was also confirmed by clear laboratory finding(s), such as increased C-reactive protein level or increased peripheral blood leukocyte count, and radiographic finding(s) such as pneumonia. Because at least two physicians had participated in the evaluation and diagnosed the occurrence of an AE from the beginning of the prospective study, the concordant rate for each exacerbation between the two physicians was almost 100%.

According to the information on the relationship between a particular SNP and the frequency of AEs, which was obtained in the retrospective study, we prospectively followed the same 276 male patients with COPD from January 1, 2003, to July 1, 2005 (30 mo). In the prospective study, we evaluated the relationship between the same SNP and the severity of the AE (endpoint: death due to the AE), instead of AE frequency.

Throughout the retrospective and prospective cohort studies, the investigators were blinded as to association of SNP allele(s) with the frequency and the severity of AEs.

Nuclear Extraction and the Electromobility Shift Assay
A monocyte cell line, THP-1 cells, was maintained in RPMI 1640 supplemented with 10% fetal bovine serum. Human recombinant IL-6 (10 ng/ml) and IFN-{gamma} (400 U/ml) were added to the medium at a cell concentration of 1.0 x 106/ml. After 6 h of the stimulation, cells were harvested and nuclear extracts were prepared by a modified method described previously (52). Oligonucleotides for electromobility shift assay (EMSA) were constructed according to the Transfac Professional database, as follows (Tables 2 and 3): CCL1-SNP3-(CCAAT/enhancer binding protein beta [C/EBPbeta])-A allele-sense: 5'-ATTGGAGTAGCTTTCACAAACCAGTCAGTAT-3', CCL1-SNP3-C/EBPbeta-A allele-antisense: 5'-ATACTGACTGGTTTGTGAAAGCTACTCCAAT-3', CCL1-SNP3-C/EBPbeta-B allele-sense: 5'-ATTGGAGTAGCTTTCACATACCAGTCAGTAT–3', CCL1-SNP3-C/EBPbeta-B allele-antisense: 5'-ATACTGACTGGTATGTGAAAGCTACTCCAAT–3'. Nuclear protein (5 µg) was incubated with labeled probes for 20 min at room temperature. The mixture was electrophoretically separated on a polyacrylamide gel and transferred to a nylon membrane. Detection of the bands was performed using a LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Inc., Rockford, IL) according to the manufacturer's instructions. Band specificity was determined by competition experiments with addition of an excess amount of unlabeled consensus oligonucleotides for C/EBPbeta, which were added to nuclear extracts before the labeled probes. A supershift assay for C/EBPbeta proteins was conducted with an anti-C/EBPbeta antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The nuclear extracts were incubated with anti-C/EBPbeta antibody (4 µg) for 1 h at 4°C before the labeled probes were added.

Statistical Analysis
The Hardy-Weinberg equilibrium was evaluated using a {chi}2 test (with p > 0.05). The association between the genotypes and the frequency of AEs in the retrospective study was evaluated using the Fisher's exact test and logistic regression analysis. We applied both dominant and recessive genetic models in these statistical analyses. Logistic regression analysis was adjusted for age, smoking index, body mass index (BMI), and lung functional index (%FEV1.0 [%predicted value]), because they are possible modifiers and we need to compensate for the effects of these modifiers in multivariate analyses. A Kruskal-Wallis test (nonparametric analysis of variance) was used to compare the difference of the average values of the ranks of the frequency of AEs in different genotype groups, according to each SNP identified in the retrospective study during the 2 yr of the study. LD coefficient (D') values among these SNPs were calculated using Haploview software version 3.31 (53, 54). The cutoff index of D' value in the software was 0.8. The survival from AEs in the prospective cohort study was analyzed in a univariate analysis using the Kaplan-Meier method and log-rank test during the 30-mo follow-up period. We also assessed the survival from AEs using a Cox proportional hazards model adjusted for age, BMI, and lung function index as covariates that are possible contributors of overall mortality (55). Results were expressed as mean ± SD. p < 0.05 was considered significant. All of the data analysis was performed using SPSS version 12.0.1J (SPSS, Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Association Analysis in the Retrospective Study
For the initial screening of the four SNPs we selected, we first compared our patients with COPD who had experienced AEs for two or more than two times (n = 73; phenotype C group: incidence of AEs, 4.27 ± 5.41 times) with those who had not experienced obvious AEs (n = 123; phenotype c group) in the retrospective study during the past 2 yr (Table 4). Patients of group C showed significantly lower BMI (kg/m2; 19.9 ± 3.3 vs. 20.9 ± 3.2, p < 0.05) and %FEV1.0 (%predicted; 43.4 ± 22.6 vs. 49.8 ± 20.4, p < 0.01) than those of group c, respectively. Our analyses of Fisher's exact test (odds ratio [OR], 2.70; 95% confidence interval [CI], 1.36–5.36; p = 0.004) and logistic regression (OR, 3.06; 95% CI, 1.46–6.41; p = 0.003) showed that SNP3 (National Center for Biotechnology Information [NCBI] SNP reference: rs2282691) in the CCL1 gene, encoding a chemotactic factor for a series of leukocytes including monocytes/macrophages (33–39), was significantly associated with the frequency of AEs in the dominant model (Table 5).


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  		TABLE 4. STUDY POPULATION FOR THE INITIAL SCREENING OF THE FOUR SINGLE NUCLEOTIDE POLYMORPHISMS IN CCL GENES

 

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  		TABLE 5. FISHER'S EXACT TEST AND LOGISTIC ANALYSIS OF THE RETROSPECTIVE STUDY IN EACH SINGLE NUCLEOTIDE POLYMORPHISM EXAMINED IN THE STUDY

 
Because exacerbation frequency increases with disease severity, which is determined by %FEV1.0 (%predicted) (32), and low BMI, which is a clinical indicator for poor prognosis (55–58), we conducted multivariate analysis to exclude the possibility that low lung functional index and BMI are acting as the confounding variables. As shown in Table 6, CCL1-SNP3, in a dominant fashion (AA+AT vs. TT), remained significant (OR, 2.82; 95% CI, 1.39–5.71; p = 0.004) regarding a predictor differentiating group C from group c. In contrast, both BMI and %FEV1.0 (%predicted) were not significant risk factors of AEs in this analysis.


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  		TABLE 6. MULTIVARIATE ANALYSIS OF PREDICTORS DIFFERENTIATING GROUP C FROM GROUP c

 
Association Analysis in the Retrospective Cohort Study
We then categorized by the incidence of AEs in the retrospective study and applied a Kruskal-Wallis test to test the difference in average values of AE frequency in three genotype groups for each SNP. In this analysis, four patients were excluded for lack of precise information on the incidence of AEs, and two patients were also excluded for recurrent low signals in genotyping SNP1. We detected SNP3 in the CCL1 gene, with a significant p value (p = 0.006; Table 7, part A), in which the risk-conferring allele is the "A" allele under the dominant model (p = 0.003; Table 7, part B). There was no significant association under the recessive model (Table 7, part C), and hence the number of AEs became significantly larger (Table 7, parts B and C). These results were in agreement with those of Fisher's exact test and logistic regression analysis. In relation to these results, D' values, including SNP3 in the CCL1 gene, were calculated. D' values among SNP1–3, SNP2–3, and SNP3–4 were 0.275, 0.254, and 0.000, respectively. This result indicates that there is no LD between SNP3 and other SNPs in this study.


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  		TABLE 7. KRUSKAL-WALLIS TEST OF THE RETROSPECTIVE COHORT STUDY FOR TESTING THE DIFFERENCE IN THE AVERAGE VALUES OF THE INFECTION RANKS IN DIFFERENT GENOTYPES IN EACH SINGLE NUCLEOTIDE POLYMORPHISM EXAMINED IN THE STUDY USING THE WHOLE PATIENTS

 
Overall, the frequency for SNP3 A and T alleles was 0.456 and 0.544, respectively, and the Hardy-Weinberg equilibrium was 0.365 (p = 0.546, {chi}2 test).

Kaplan-Meier Analysis with Log-Rank Test of the Prospective Cohort Study
The purpose of the prospective study was to evaluate the relationship between the genotype of SNP3 in CCL1 gene and, unlike the retrospective study, the severity (endpoint: death) of the AEs. No patients dropped out or were eliminated except in cases of death. According to the results in the retrospective cohort study (Table 7), we applied the Kaplan-Meier method with the log-rank test for testing the differences in trends in both the genotype and A allele–dominant survival rate curves. This is based on the fact that the follow-up study had been performed for only 30 mo, not long enough to see patients' long-term outcome (Table 8). By analyzing 30-mo follow-up data with the Kaplan-Meier method, we found that the A allele, which is a significant risk allele for the severity of AEs in COPD, influenced the death (mortality) due to AEs in this prospective study. Carriage of the A allele decreased the average survival period because of an increased risk of death due to AEs. We have also found that the homozygous A genotype shows more significant association than do heterozygous A genotypes. Although the heterozygous genotype compared with the "T" homozygous genotype does not reach significance (p = 0.06), it shows the significant association in both dominant and recessive (log-rank statistic, 4.43; p = 0.0353) models. For these reasons, the heterozygous genotype can also be considered to be the risk type for severity (mortality) of AEs in COPD, and a typical "gene–dosage effect" of the risk allele A to the negative impact on their survival is shown in Figure 2.


Figure 2
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  		Figure 2. Kaplan-Meier method, with log-rank test, for testing the differences in trends in both the genotype- and A allele–dominant survival rate curves of the prospective cohort study during the 30 mo according to presence of SNP3 (NCBI SNP reference, rs2282691) in the CCL1 gene. (A) Three survival rate curves according to genotype. (B) Two survival rate curves according to a dominant model. See Table 8 for statistical results. The cross marks on each survival rate curve indicate the censored observations due to death except for death caused by acute exacerbations (AEs).

 

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  		TABLE 8. KAPLAN-MEIER METHOD, WITH LOG-RANK TEST, FOR TESTING THE DIFFERENCES IN TRENDS IN BOTH THE GENOTYPE AND "A" ALLELE–DOMINANT SURVIVAL RATE CURVES OF THE 30-MONTH PROSPECTIVE COHORT STUDY ACCORDING TO THE PRESENCE OF SNP3* IN THE CCL1 GENE

 
Cox Proportional Hazards Regression Analysis in the Prospective Cohort Study
We next analyzed 30-mo follow-up data with the Cox proportional hazards regression method. We found the proportional hazards trend for SNP3 in the CCL1 gene, and that the Cox proportional hazards regression model can be effectively applied to our results (Table 9). In our two models, age is considered to be the risk factor for the severity (mortality) of AEs (p = 0.041 and 0.038, respectively). In the early stages of the prospective study, nutrition (BMI) and lung functional index (%FEV1.0 – %predicted value) did not show much significance. However, we cannot reject the possibility that these factors are involved in the severity and mortality of AEs in COPD (55). The summary of the analysis is shown in Figure 3, which essentially replicates the results of the Kaplan-Meier analysis in this study. A typical gene–dosage effect was also observed.


Figure 3
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  		Figure 3. Cox proportional hazards regression method for testing the differences in both the genotype- and A allele–dominant survival rate curves of the prospective cohort study during the 30 mo according to presence of SNP3 (NCBI SNP reference, rs2282691) in the CCL1 gene. (A) Three survival rate curves according to genotype. (B) Two survival rate curves according to a dominant model. See Table 9 for statistical results.

 

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  		TABLE 9. COX PROPORTIONAL HAZARDS REGRESSION METHOD FOR TESTING THE DIFFERENCES IN BOTH THE GENOTYPE AND "A" ALLELE–DOMINANT SURVIVAL RATE CURVES OF THE 30-MONTH PROSPECTIVE COHORT STUDY ACCORDING TO THE PRESENCE OF SNP3* IN THE CCL1 GENE

 
In the prospective study, 47 patients died. Among them, 23 patients (48.9%) died of AEs. Sputum samples of 12 of these patients yielded a positive culture of a possible pathogen, whereas the other 11 samples yielded nonspecific culture growth. The other causes of death were as follows: malignant diseases for 15 patients, cardiovascular complications for 6 patients, respiratory failure for 2 patients, and accident for 1 patient.

EMSA
To assign the function of SNP3, which resides in intron 2, we sought for potential transcription factor binding site surrounding SNP3 using the Transfac Professional database (Biobase Biological Databases) (45–48). We found that the sequence with the A allele did not show any match but the T allele was shown to be a putative transcriptional factor binding site for C/EBPbeta and transcription factor poly(C)-binding protein 2 (TFCP2), with Transfac scores of 89 and 87, respectively (Tables 2 and 3). We therefore determined whether the sequence surrounding SNP3 was capable of binding to a protein using EMSA. We excluded TFCP2 as a potential transcriptional factor in this experiment, because it is a major factor in the regulation of globin gene expression in human erythroid cells and is not related to inflammation or innate immunity functions (59). Nuclear extracts were prepared from the monocyte cell line THP-1 after stimulation and EMSA was performed using oligonucleotides SNP3-A and SNP3-T, which correspond to sequences for A and T alleles in the CCL1 gene. A nuclear extract–oligonucleotide complex gave rise to shifted bands with SNP3-T but not with SNP3-A (Figure 4, lanes 2 and 5, respectively). The specificity of the band was confirmed by cold competition (Figure 4, lane 4). In addition, this complex was supershifted by adding anti-C/EBPbeta antibody (Figure 4, lane 3). This result indicates that SNP3-T but not SNP3-A can bind to C/EBPbeta.


Figure 4
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  		Figure 4. Effect of the difference between the oligonucleotides SNP3-A and SNP3-T, which correspond to sequences for A and T alleles in the CCL1 gene, on the protein–DNA complex formation demonstrated by the electromobility shift assay. Lane 1: Oligonucleotide probe of C/EBPbeta with reference to SNP3-T without nuclear extract was applied. Free probe was detected far below a protein-DNA complex, and cannot be observed in this figure. Lane 2: A nuclear extract–oligonucleotide probe of C/EBPbeta with reference to SNP3-T was applied. A protein–DNA complex is indicated by an asterisk. Lane 3: A nuclear extract–oligonucleotide probe of C/EBPbeta with reference to SNP3-T, incubated with anti-C/EBPbeta antibody, was applied. Supershifted band is indicated by an arrow. Lane 4: Band specificity was confirmed by a competition experiment with a molar excess of unlabeled (cold) oligonucleotide probe of C/EBPbeta with reference to SNP3-T. Lane 5: a nuclear extract–oligonucleotide probe of C/EBPbeta with reference to SNP3-A was applied.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study showed that, in COPD, individual susceptibility to AEs, indicated by the association analyses between the SNPs and the frequency and severity of AEs, may be attributed, at least in part, to the genetic variance of CCL1, a gene encoding a chemotactic factor for a series of leukocytes including monocytes/macrophages (33–39). The marked heterogeneity in the host defense mechanisms against the offending pathogens and in the degree of the inflammatory response during AEs (7, 11–15) may partially be explained by this variation. Initially, we prioritized the SNPs in CXCL8 and CXCL5 genes, both of which are representative neutrophil chemoattractants (CXCL subfamily) and which play an important role in AEs of severe COPD (23), because a considerable number of AEs (80.4%) occurred in group C patients (Table 4; n = 73; %FEV1.0 [%predicted], 43.4 ± 22.6%), whose classification of severity was mostly severe (60). If findings of the association analyses in CXCL8 and CXCL5 genes proved to be negative, our second-tier choice was to analyze the SNPs in CCL11, CCL1 and CCL5 genes, all of which are important eosinophil chemoattractants (CCL subfamily), and an "allergic" profile of inflammation is one of the characteristic features in exacerbations of mild COPD (21, 22). However, at the beginning of this study, there were no SNPs reported in the CXCL5 gene, and there were three SNPs in the CXCL8 gene, whose allelic frequencies were unknown in the Japanese population, found by searching the nucleic acid database. Therefore, we conducted association analyses of the SNPs in CCL11, CCL1, and CCL5 with the frequency and severity of AEs in a cohort of 276 male patients with COPD in the retrospective and prospective studies.

According to this SNP selection, a single SNP (SNP3: NCBI SNP reference, rs2282691) in the CCL1 gene, encoding a chemotactic factor for a series of leukocytes including monocytes/macrophages (33–39), showed a significant association with the frequency of AEs. This was identified in Fisher's exact test, logistic regression analysis, and a Kruskal-Wallis test in the retrospective cohort study, and was confirmed in the prospective cohort study with the severity (endpoint, death) of AEs with the Kaplan-Meier method with a log-rank test and Cox proportional hazards regression method. In addition, SNP3-T was located within the putative transcriptional factor binding sites of C/EBPbeta, as shown by a high Transfac score, whereas none of the putative transcriptional factor binding sites were detected in SNP3-A (Tables 3 and 2, respectively) (45–48). Experiments using EMSA clearly demonstrated that this is the case (Figure 4).

CCL1 is one of the genes that reside in a CCL subfamily chemokine gene cluster on chromosome 17q11.2-q12. According to the NCBI built 34, CCL1 is not the only chemokine-related gene located beside CCL11 and CCL5 genes. The CCL8 gene with two SNPs and the CCL13 gene with three SNPs are located beside CCL11 and CCL5 genes. The SNPs beside CCL11 and CCL5 genes were required to analyze local LD (40–42, 44). However, among these SNPs, only one SNP in the CCL1 gene (SNP3) had an appropriate minor allele frequency (MAF; MAF of 0.48 in the Japanese population), whereas the MAFs with other SNPs in this region in the Japanese population were unknown. In general, it is favorable for the MAF to be more than 10 to 15% for an SNP analysis in many populations (61, 62). Furthermore, it has been demonstrated that the axis of CCL1 and its specific receptor (expressed on both monocytes/macrophages and Th2 cells) link adaptive and innate immune functions, which play an important role in the initiation and amplification of allergic inflammation through the activation of Th2 cells, such as Th2-polarized T lymphocytes and NK cells, and antigen-elicited eosinophils (34–39). An allergic profile of inflammation is one of the characteristic features in exacerbations of mild COPD (21, 22). For these reasons, we conducted SNP genotyping and statistical analysis of SNP3 in the CCL1 gene in relation to the frequency and severity of AEs, together with SNP1 and SNP2 in the CCL11 gene and SNP4 in the CCL5 gene, both of which are representative eosinophil chemoattractants and are strongly expressed in the surface epithelium and subepithelial lymphomononuclear cells in exacerbations of mild COPD (19–22).

Although SNP3-A is a risk allele, carriage of SNP3-T was relatively associated with protection from AEs throughout the study. There are two reasons for this observation. First, the number of AEs became significantly smaller in the SNP3-T homozygous genotype in the retrospective cohort study (Table 7); and second, the SNP3-T homozygous genotype as well as the heterozygous genotype had an increased average survival rate due to the relative protection conferred by this allele from dying of severe AEs (Table 8). This result in the prospective cohort study showed a typical gene–dosage effect, although some of the data did not reach statistical significance (Tables 8 and 9; Figures 2 and 3). As shown in Figure 4, oligonucleotide probes of C/EBPbeta with reference to SNP3-T, but not SNP3-A, were able to bind to C/EBPbeta. This was in agreement with the prediction by the Transfac Professional database (Tables 3 and 2, respectively). This result leads to evidence for the functionality of SNP3 and suggests its clinical relevance by speculating on the nature and behavior of C/EBPbeta molecule in the lungs.

C/EBPs are a family of leucine zipper transcription factors involved in the regulation of various aspects of cellular differentiation and linked processes, such as proliferation, apoptosis, and gene expression, in multiple tissues (63). C/EBPs are also central to inflammatory response and the activation of early inflammatory genes and defense mechanisms against infection, all of which are important for innate immunity functions (63). Among three isoforms expressed in the lung epithelium—namely, C/EBP{alpha}, C/EBPbeta, and C/EBP{delta} (64)—C/EBPbeta is the dominant form of C/EBP in the airway epithelium (65). The cardinal role of C/EBPbeta in the lungs is to react and up-regulate a set of C/EBPbeta-regulated genes, including the CCL1 gene, as a key transcriptional factor of lung-specific acute-phase molecules in response to different stimuli, such as acute lung injury or infection, thus leading to the subsequent activation of the innate immune system and inflammatory response (63–65). In fact, C/EBPbeta–/– mice are extremely susceptible to infections with microorganisms such as Candida albicans and Listeria monocytogenes, mainly as a result of the defects of the C/EBPbeta-specific defense mechanisms in monocytes/macrophages (63). On the other hand, our retrospective and prospective data demonstrated the relative protection from AEs of SNP3-T compared with SNP3-A in the CCL1 gene. Collectively, these findings suggest that SNP3-A in the CCL1 gene predisposes patients with COPD to pulmonary infections, compared with SNP3-T, due to the different binding capacity of C/EBPbeta, as shown in Figure 4. This leads to speculation that the genotype-dependent expression of CCL1 protein, a chemotactic factor for a series of leukocytes including monocytes/macrophages, secreted mainly by activated T lymphocytes (33–39), in the acute phase of inflammatory responses caused by infectious pathogens may influence the appropriate formation of the chemokine/receptor network against offending pathogens (19, 20), and consequently may be associated with individual susceptibility to AEs. In support of this notion, a massive infiltration of subepithelial lymphomononuclear cells in the bronchial mucosa is observed even in the stable state in patients with COPD (21–23).

Although exacerbation frequency increases with disease severity, which is determined by %FEV1.0 (%predicted) (32), our multivariate analysis in the retrospective study suggested that %FEV1.0 (%predicted) was not a significant risk factor for AEs (Table 6; p = 0.115). This is partially explained by the influence of the other variables, CCL1-SNP3 in a dominant fashion (AA+AT vs. TT), which showed much significance in this analysis (Table 6; p = 0.004). However, on the basis of the London study, which is a prospective, accurate (daily peak flow and/or spirometry and symptoms on individual diary cards) study of patients with COPD, the exacerbation frequency was also associated with %FEV1.0 (%predicted), and the annual exacerbation frequency was higher than that reported in Table 4 in our data for similar values of %FEV1.0 (%predicted) (66). In fact, in the present study, the average incidence of AEs per patient per year in the retrospective phase of 2 yr was 0.71, which was much lower in frequency than in previous reports (2.5–3.0 [10, 16]). This discrepancy can be explained, because the previous studies are prospective cohort studies and some AEs were assessed from the patients' daily diary card data each month to clarify the unreported exacerbations (10, 16). In contrast, in the present study, we assessed the frequency of AEs by assessing the medical records of each individual retrospectively with two independent pulmonary physicians. Moreover, the evidence of AEs had to be confirmed by clear laboratory finding(s), such as increased C-reactive protein level or increased peripheral blood leukocyte count, and radiographic finding(s), such as pneumonia; and concordance for diagnosis between the two physicians was required (51). We believe this stringency for diagnosis in our study was indispensable to avoid bias from the patients and to increase objective evaluation for the physicians. In support of this notion, the relative but not absolute numbers of the frequency of AEs (Table 4) are considered to be correct, because of the significantly lower values of %FEV1.0 (%predicted) and BMI in group C compared with those in group c, both of which are associated with disease severity and exacerbation frequency increases (group C > group c) with disease severity (32, 55–58, 66).

In the present study, the all-cause mortality rate per year in the 30-mo prospective phase was 6.8% (3.3% in a control population), and the initial mean %FEV1.0 (%predicted) and age were 47.0% and 73.7 yr, respectively. This is similar to previous reports (66–68). In the London study, the all-cause mortality rate was 5.7% per year and the initial median %FEV1.0 (%predicted) and age were 38.4% and 68.4 yr, respectively (66). In the Tucson study, the all-cause mortality rate was 5.3% per year and the initial mean %FEV1.0 (%predicted) and age were 47.1% and 64.6 yr, respectively (67). In the IPPB trial, the all-cause mortality rate was 7.7% per year and the initial mean %FEV1.0 (%predicted) and age were 36.1% and 60.9 yr, respectively (68). In addition, in the Tucson study, the primary cause of death was a respiratory disorder (considered to be an AE plus respiratory failure) in 52% of those who died(67). In the present study, mortality due to an AE plus respiratory failure was 53% among overall mortality. Furthermore, in the IPPB trial, patient initial age and %FEV1.0 (%predicted) were the best predictors of mortality (68). In the present study, Cox proportional hazards regression analysis indicated that patient initial age and %FEV1.0 (%predicted) were also the predictors of mortality despite the influence of the SNP3 genotype, which showed more pronounced significance in both the genotype and A allele–dominant models (Table 9; AA vs. TT, p = 0.023; AA+AT vs. TT, p = 0.036), although %FEV1.0 (%predicted) did not reach statistical significance in the early stages of follow-up (Table 9; p = 0.073 and 0.065, respectively). Taken together, the results of our prospective cohort study are not in conflict with the expected values from the literature (66–68).

The difference in binding capacity of the C/EBPbeta between SNP3-T and SNP3-A in the CCL1 gene does not necessarily provide enough evidence that SNP3 actually affects transcriptional regulatory activity in the CCL1 gene. We, therefore, measured the serum levels of CCL1 with an enzyme-linked immunosorbent assay in 91 patients enrolled in this study after follow-up. There was a genotype-dependent difference in the average values of the serum CCL1 among three genotypes (AA: n = 17, 2.45 ± 0.78 pg/ml; AT: n = 45, 2.92 ± 1.32 pg/ml; TT: n = 29, 2.96 ± 2.33 pg/ml), although there was no statistical significance. Lack of significance may be partially attributed to a survivor effect and/or to the fact that serum levels of CCL1 were measured under nonstimulated conditions. Functional analysis, such as reporter-gene assay and measurement of CCL1 levels produced by peripheral blood leukocytes under stimulated conditions, will be necessary to confirm the transcriptional regulatory activity of SNP3 in the CCL1 gene (69–71).

In summary, we identified a single SNP (SNP3; NCBI SNP reference, rs2282691) in the CCL1 gene, encoding a chemotactic factor for a series of leukocytes including monocytes/macrophages (33–39), with significant relation to the frequency and severity of AEs in retrospective and prospective studies of a cohort of 276 male patients with COPD. Carriage of SNP3-A, acting as the risk allele, predisposes the patients with COPD to AEs. In contrast, carriage of the SNP3-T is associated with relative protection from AEs. This unique phenomenon appears to stem from the binding capacity of C/EBPbeta, a key transcriptional factor of lung-specific acute-phase protein in response to acute pulmonary infections, to its particular transcriptional factor binding sites of the CCL1 gene. We speculate that the difference of the genotype-dependent expression of CCL1 protein may lead to the alteration of the subsequent activation of the innate immune system and inflammatory response, through the altered formation of the chemokine/receptor network against acute pulmonary infection. Although functional analysis remains to be elucidated, this study provides an insight toward better understanding of the mechanisms of individual susceptibility to AEs, which may ultimately lead to creating more effective therapeutic or prophylactic strategies in the future.


   Acknowledgments
 
In addition to the authors, the following investigators and Japanese institutions participated in the study: Shinoda General Hospital, Yamagata—H. Atsumi; Okitama General Hospital, Kawanishi—M. Inage and S. Kato; Japan Sea Hospital, Sakata—H. Saito; Saisei Hospital, Yamagata—H. Takeda; Shinjo Prefectural Hospital, Shinjo—Y. Katagiri and K. Otake; Yonezawa City Hospital, Yonezawa—H. Yuki; Sagae City Hospital, Sagae—M. Sato; Saiseikan Hospital, Yamagata—J. Sato and K. Iwabuchi; Kahoku Prefectural Hospital, Kahoku—H. Suzuki; Tohoku Central Hospital, Yamagata—T. Sayama and K. Shida; Kaminoyama Nagaoka Clinic, Kaminoyama—M. Nagaoka; Yakuwa Clinic, Murayama—N. Yakuwa; Kudo Clinic, Kahoku—Y. Kudo: Sasai Clinic, Yonezawa—Y. Sasai. The authors also thank Masayuki Saito, Ph.D., and Sayumi Toriyama, Ph.D. (HuBit Genomix, Inc., Tokyo, Japan), for their technical discussion.


   FOOTNOTES
 
Supported by a grant-in-aid from the 21st Century Center of Excellence program of the Japan Society for the Promotion of Science, and in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (nos. 15790407, 17590778, 14770268, 16590733, 15590796, 17590779, 17590702, and 14570635).

Originally Published in Press as DOI: 10.1164/rccm.200603-4430C on July 24, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 30, 2006; accepted in final form July 24, 2006


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