Ljungman et al. Although FGB rs1800790 displayed an interaction effect with CO on IL-6 levels, we found no effect of the polymorphism on IL-6 in these patients when not taking air pollution into account. A previous study of the same population (Jacquemin et al. 2008) investigating the effect of fibrinogen poly- morphisms on fibrinogen plasma levels found no interaction effect with high levels of IL-6 on fibrinogen levels for this polymorphism, in contrast to FGA rs2070011. However, a follow-up study (Peters et al. 2009) showed that FGB rs1800790 modified the effect of a 5-day average exposure to ambient PM on cir- culating fibrinogen levels. The production of fibrinogen is stimulated by IL-6, but there is also evidence both in vivo (Szaba and Smiley 2002) and in vitro (Robson et al. 1994) suggesting a positive feedback mechanism whereby fibrinogen products in turn stimulate IL-6 production and release from monocytes. Although this might suggest that the increased IL-6 levels seen in MI survivors with a minor allelic genotype of FGB rs1800790 are medi- ated via increased fibrinogen levels, the effects on IL-6 levels occurred within 12 hr rather than after 5 days. In this study, subjects with major alleles of IL6 rs2069832 and rs2069845 had lower IL-6 concentrations than did those with minor alleles, but they seemed to react more strongly to CO exposure than did minor allele holders. The reason for this pattern is unclear. Our previous study (Ljungman et al. 2008) revealed a tendency of a greater within-individual variability for major allele holders compared with minor allele holders. Taken together, this may indicate that major allele holders are more likely to respond with inflammation to stimuli such as air pollution but from a lower basal level of IL-6. Air pollution has been associated in epide- miologic studies with the onset of MIs in both previously healthy subjects and cardiac patients (Peters et al. 2001; Zanobetti and Schwartz 2005). In addition, human experimental (Mills et al. 2007) and epidemiologic (Chuang et al. 2008) studies have demonstrated an effect of air pollution on ST-segment changes in electrocardiograms indicating the triggering of acute ischemia. The gene–environment interaction observed for these two SNPs in our study emphasizes that certain genetic sub- groups of cardiac patients may react more strongly toward air pollution than do others. Similar genetic interaction with air pollutants has been seen in human and animal models for lung disease (Kleeberger 2003; London 2007) and oxidative stress (Romieu et al. 2006; Schwartz et al. 2005). Although both the IL6 and FGB genes are probable candidates affect- ing the levels of the known cardiovascular biomarker IL-6, the actual polymorphisms responsible for the observed effect modification 1378 may be other strongly correlated SNPs or com- binations of SNPs. Indeed, in our population, IL6 rs2069832 has very high LD with IL6 rs1800795, a functional SNP located in the promoter region. The reason for focusing on IL6 rs2069832 instead of IL6 rs1800795 was that the overall pooled effects of IL6 rs1800795 on IL-6 levels showed slightly more hetero- geneity across cities (Ljungman et al. 2008). To confirm the effects of the studied poly- morphisms, functional analyses are required, and as our analyses suggest, the nature of pos- sible gene–gene interaction effects in combina- tions of SNPs possibly in different genes are of interest. In order to verify whether these data have any prognostic significance, follow-up of clinical ischemic events has to be performed. Strengths and limitations. We conducted this study with data from six European cities, thereby providing six independent samples for testing gene–environment interactions. We focus here on genes that, applying a meta- analytical approach across all cities, were asso- ciated with gene–environment interactions. The different cities, however, reflect not only a large variation of air pollution expo- sure, which was desirable, but also other dif- ferences possibly confounding our results. To control for this variation, we applied a two-step approach, starting with city-specific models and thereafter pooling the results and checking for evidence of heterogeneity. This test, however, suffers from low power, and the interaction effects showed some dispar- ity across cities. We found clearer effects in Athens and Barcelona than in Helsinki or Stockholm. The southern European cities showed the highest mean levels and the great- est variability in CO levels, whereas Stockholm and Helsinki showed very little variation and considerably lower mean levels. A sensitivity analysis excluding Athens did reduce the effect estimates for CO and increased the p-values of interaction, whereas results for NO2 and PM2.5 were essentially unchanged (data not shown). However, we present here a pooled analysis of six independently conducted stud- ies based on a common protocol. Therefore, we regard the a priori planned analyses as valid, which include all cities, and especially those with the large exposure contrasts and therefore more power. By and large, the effects for CO were corroborated by those for NO2. The remaining differences might be attributed to possible differences in measurement error for the pollutants, as well as possible differ- ences in how representative the fixed monitors are of urban background levels of the various pollutants. The low-risk allele showed point estimates below zero for the IL6 SNP and the combined SNPs in relation to increased CO, for which we have no clear explanation, but because we found no such tendency for the same genotypes in relation to NO2, this may be a chance finding. The temporal relation- ship between exposure and effect within 12 hr generally fits the expected rise and fall of IL-6 response in the bloodstream. A fair amount of measurement error was seen for IL-6, but we expect that this would not be associated with air pollution levels and therefore would lead to nondifferential misclassification, potentially attenuating our results. We had expected a stronger main effect of CO and NO2 on IL-6 levels, but because air pollution is strongly linked to cardiovas- cular mortality, survivors of MI—although at high absolute risk for future cardiovascular events—may be less susceptible to the effects of air pollution in a relative sense than the general population. Nevertheless, our results indicate that groups of these individuals iden- tified by specific genotypes are at increased risk of a systemic inflammatory response in association with exposure to continuous ambient air pollution. Should this increase in IL-6 be confirmed to have prognostic impli- cations, these individuals could be identified and protected with counseling such as avoid- ance of traffic or going outdoors at periods of high risk, for example, within the first month after an MI or during highly polluted days. Furthermore, they might be helped by more rigorous medication. Conclusion The results support our hypothesis that the effect of air pollution on inflammation may be stronger in certain genetic subpopulations of cardiac patients. Polymorphisms in the IL6 genes modified the IL-6 response when exposed to increased air pollution in the 6–11 hr period preceding sampling, consistent with biological expectation. The role of FGB rs1800790 also seemed important, although the rapid effect was unexpected. Combining these polymor- phisms also showed a gene–gene interaction, possibly implicating that one-sixth of the pop- ulation of MI survivors is at particular risk for effects of air pollution. Whether these differ- ences in air-pollution–mediated inflammation have any prognostic importance remains to be clarified, as well as the possible role of these polymorphisms in air pollution effects on ear- lier stages of cardiovascular morbidity. Ref Rencs Anonymous. 2000. Myocardial infarction redefined—a consen- sus document of the Joint European Society of Cardiology/ American College of Cardiology Committee for the redefini- tion of myocardial infarction. Eur Heart J 21(18):1502–1513. Bennermo M, Held C, Green F, Strandberg LE, Ericsson CG, Hansson LO, et al. 2004. Prognostic value of plasma inter- leukin-6 concentrations and the –174 G > C and –572 G > C promoter polymorphisms of the interleukin-6 gene in patients with acute myocardial infarction treated with thrombolysis. Atherosclerosis 174(1):157–163. Brook RD, Franklin B, Cascio W, Hong Y, Howard G, Lipsett M, et al. 2004. Air pollution and cardiovascular disease: a state- ment for healthcare professionals from the Expert Panel on ovlume 117 | number 9 | September 2009 • Environmental Health Perspectives