Ljungman et al. We found no evidence of deviation from the additive model for these SNPs (p-value for heterozygote indicator variable > 0.05). Overall effects of air pollution on IL-6 levels. Elevated concentrations of CO averaged during the 0–24 hr preceding blood sampling were not significantly associated with increased plasma IL-6 levels (Table 3), but elevated con- centrations during 6–11 hr intervals preced- ing sampling were associated (2.2% change of overall mean IL-6; 95% CI, 0.3–4.2%). For other pollutants, NO2 levels averaged over the 0–24 hr preceding blood sampling showed associations with an increase in plasma IL-6 PM2.5 PM10 Table 3. Effect of 24-hr interquartile range increase in air pollution on IL-6 plasma levels. Air pollutant response Interquartile Change of IL-6a (% of overall mean without genetic covariate range increase per interquartile range increase) CO 0.34 mg/m3 0.8 (–0.5 to 2.1) 0.22 NO2 15.9 µg/m3 1.7 (0.2 to 3.4) 0.03 PNC 11,852/cm3 1.9 (–0.2 to 4.0) 0.07 11.0 µg/m3 0.6 (–0.8 to 2.0) 0.40 17.4 µg/m3 0.0 (–1.3 to 1.3) 1.0 aInverse-weighted sum of city-specific estimates and 95% CIs from linear regression using log IL-6 concentrations as outcome, with a random effect for subject, using the 24-hr average of air pollutants fitted to the time of IL-6 measure- ment and adjusting for age, city, BMI, log NT-proBNP, HDL cholesterol, systolic blood pressure, number of MIs, glyco- sylated hemoglobin, pack-years of cigarette smoking, alcohol intake, history of arrhythmias, heart failure, bronchitis, season, apparent temperature, relative humidity, and hour of visit in city-specific models. p-Value Table 4. SNP–environment interactions: association between 24-hr average of CO or NO2 and plasma IL-6 levels by genotype for the analyzed polymorphisms in the genes IL6, FGA, and FGB. Change of IL-6a (% of overall mean per interquartile range increase of pollutant) Genotypeb 1 1 1 2 2 2 1 1 1 2 2 2 1 1 1 2 2 2 1 1 1 2 2 2 p-Value p-Value p-Value p-Value CO 0.03 0.04 2.0 (0.3 to 3.6) –0.2 (–1.7 to 1.3) –2.0 (–4.7 to 0.8) 2.0 (0.3, 3.8) 0.4 (–0.9, 1.7) –1.2 (–3.4, 1.1) 1.9 (0.2 to 3.5) –0.1 (–1.5 to 1.4) –1.6 (–4.3 to 1.2) 0.31 1.0 (–0.7 to 2.7) 0.7 (0.6 to 2.0) 0.4 (–1.9 to 2.7) 0.64 NO2 3.1 (0.9 to 5.4) 1.2 (–0.5 to 3.0) –0.5 (–3.5 to 2.5) 0.09 1.8 (–0. 4 to 4.0) 1.7 (0.0 to 3.4) 1.7 (–1.5 to 4.9) 0.93 2.8 (0.5 to 5.1) 1.3 (–0.4 to3.1) 0.2 (–2.6 to 3.2) 0.26 2.1 (–0.1 to 4.3) 1.6 (–0.1 to 3.3) 1.1 (–1.8 to 4.1) 0.64 SNP IL6 rs2069832 IL6 rs2069840 IL6 rs2069845 FGA rs2070011 )15 R -6 Ir L e10 I p in n c e e a 5 ren llm f ra 0 e f i e D vo (%–5 1376 FGB rs1800790 p-Value 1 1 –0.2 (–1.8 to 1.3) 1.0 (–0.9 to 2.9) 1 2 2.1 (0.4 to 3.8) 2.8 (0.6 to 5.0) 2 2 4.5 (1.1 to 8.0) 4.4 (0.1 to 8.9) 0.02 0.19 aInverse-variance weighted sum of city-specific estimates and 95% CIs from linear regression using IL-6 concentrations as outcome and an additive genetic model for the genotype, with a random effect by subject, using the 24-hr average of CO or NO2 fitting to the time of IL-6 measurement, including a genotype–CO interaction adjusted for age, city, BMI, log NT-proBNP, HDL cholesterol, systolic blood pressure, number of MIs, glycosylated hemoglobin, pack-years of cigarette smoking, alcohol intake, history of arrhythmias, heart failure, bronchitis, season, apparent temperature, relative humidity, and hour of visit in city-specific models. Interquartile range increase: CO, 0.34 mg/m3; NO2, 15.9 µg/m3. b1, major allele; 2, minor allele. IL6 rs20698322 FGB rs1800790 Q Q p = 0.03 p = 0.09 p = 0.45 p = 0.23 p = 0.76 Major allelic homozygote 1 1 Heterozygote 1 2 Minor allelic homozygote 2 2 * * )15 R -6 Ir L e10 I p in n c e e a 5 ren llm f ra 0 e f i e D vo (%–5 p = 0.02 p = 0.19 p = 0.89 p = 0.03 p = 0.07 CO NO2 PNC PM2.5 PM10 CO NO2 PNC PM2.5 PM10 Figure 1. Modification by IL6 rs2069832 (A) and FGB rs1800790 (B) genotypes of IL-6 response to increased 24-hr average ambient CO, NO2, ultrafine PM (PNC), and PM2.5 immediately preceding blood withdrawal (1, major allele; 2, minor allele). Error bars indicate 95% CIs; p-values are for significance of the interaction term. Interquartile ranges: CO, 34.0 mg/m3; NO2, 15.9 µg/m3; PNC, 11,852/cm3; PM2.5, 11.0 µg/m3; PM10, 17.4 µg/m3. *Heterogeneity of the city-specific effect estimates with a p-value < 0.1. (Table 3), and PNC levels showed significant associations for both 6–11 hr and 12–17 hr exposure windows preceding sampling [1.8% change (95% CI, 0.2–3.5%) and 2.6% change (95% CI, 0.8–4.5%), respectively]. Genotype modification of IL-6 response (24-hr exposure windows). Subjects with the homozygous major allele genotypes for all three IL6 polymorphisms showed larger IL-6 responses to increased CO, but only for for IL6 rs2069832 was a similar genetic inter- action seen with increased NO2 (Table 4). Subjects with the homozygote minor allele genotype of FGB rs1800790 showed both a larger and clearer effect modification for the IL-6 response to increased CO com- pared with the IL6 SNPs. The overall effect of a 0.8% increase in IL-6 per 0.34 mg/m3 increase of CO in the preceding 0–24 hr seemed to be confined to individuals carry- ing the minor allele of FGB rs1800790. In the 4% of the study sample that were homo- zygous with this allele, the corresponding CO effect on IL-6 was a 4.5% increase. We saw similar magnitudes of effect modification for NO2, but the effect modification pattern was not statistically significant. The FGA SNP did not modify the response to air pollution. These results were robust to sensitivity analysis excluding all time-invariant variables from the final model and only maintaining time trend and meteorology (data not shown). For PM pollutants, we found a simi- lar pattern of effect modification of FGB rs1800790 for increased levels of PM2.5 and PM10 during the preceding 0–24 hr, but with less precision of effect for PM10 (Figure 1). For ultrafine particles measured by PNC and for IL6 rs2069832, the pattern was less clear. When we classified different combinations of the IL6 rs2069832 and FGB rs1800790 genotypes according to number of risk alleles into low, intermediate, and high risk (Figure 2A), we found significant modifica- tion of the IL-6 response to an increase in the preceding 0–24 hr average of CO as well as both NO2 and PM2.5 (Figure 2B). Subjects with ≥ three high-risk alleles from both SNPs (154 subjects) had the largest IL-6 response. Genotype modification of IL-6 response (6-hr exposure windows). Analyses of 6-hr exposure windows during the 24 hr immedi- ately preceding blood sampling showed the clearest effect modification of the IL-6 response to CO within the three preceding 6-hr win- dows (Figure 3). We found greatest effect modification for FGB rs1800790 for increased CO during the 6–11 hr preceding sampling. The overall effect of a 2.2% increase in IL-6 per 0.64 mg/m3 increase of CO in this time window seemed again to be confined to individuals carrying the minor allele of FGB rs1800790, with a 10% (95% CI, 4.6–16%) increase in homozygotes (4% of study sample). ovlume 117 | number 9 | September 2009 • Environmental Health Perspectives