Inflammatory Cytokines and Risk of Coronary Heart Disease
Inflammatory Cytokines and Risk of Coronary Heart Disease
The current results indicate that circulating levels of several different pro-inflammatory cytokines in initially healthy people are associated with risk of CHD outcomes in an approximately log-linear manner. These associations appear to be largely independent of several conventional and emerging cardiovascular risk factors. Furthermore, updated meta-analyses reinforce the validity and generalizability of these new data, suggesting that a 1-SD higher baseline level for each of IL-6, IL-18, and TNF-α is associated with ~10–25% higher risk of non-fatal MI or CHD death. Analyses that make allowances for the considerable fluctuations that we observed of these analytes within individuals over a few years would increase the estimated size of these associations with CHD (see below). Similar associations have previously been reported for CRP and fibrinogen based on meta-analysis of individual participant data, hence we only analysed the limited data available on CRP in our primary study for making direct internal comparisons. Collectively, the current study establishes robust observational associations of several pro-inflammatory cytokines with the risk of CHD, which lends further support to the inflammation hypothesis in vascular disease. Although this does not establish causality in CHD for any of the analytes, the comprehensive evaluation of their associations with CHD should be important, given the evolving literature on cytokines as potential drug targets and gaps in translating basic scientific findings into clinical practice.
Interleukin-6 is involved in the systemic inflammatory response, but also engages in local tissue inflammation and promotes differentiation of naive T-helper cells into Th17 cells, a cell type that has been implicated in auto-immune conditions such as rheumatoid arthritis. Tumour necrosis factor-α is a pro-inflammatory cytokine, which, similar to IL-6, has been implicated in auto-immune diseases. Both IL-6 and TNF-α signalling pathways are targets of drugs (such as tocilizumab and etanercept) which are used to treat auto-immune diseases. Moreover, there have been suggestions of an increased incidence of CVD in patients with rheumatoid arthritis, and non-randomized data from biological registries suggest TNF-α blocking therapy could reduce CVD risk in these patients, although such data have well-recognized limitations. It is of interest, therefore, that two of the agents (i.e. canakinumab, a monoclonal antibody to IL-1β, and low-dose methotrexate) being tested in phase 3 trials of CVD prevention lower circulating IL-6 levels, while low-dose methotrexate therapy also lowers TNF-α levels. Furthermore, consistent with previous studies, we found circulating IL-18 levels to be positively associated with CHD incidence. In contrast, we found no significant associations of sCD40L and MMP-9 levels with CHD in the aggregate of available prospective studies, suggesting that positive findings in previous non-prospective data may have been liable to potential biases, e.g. 'reverse causality'.
Our study had several strengths. First, whereas most previous studies have concurrently considered only a few cytokines, we were able to measure simultaneously multiple pro-inflammatory cytokines in a common subset of participants in one central laboratory, facilitating uniform comparison of the observed associations across markers. Furthermore, we excluded all individuals having baseline CVD to minimize any reverse association bias. Additionally, we combined data from previously published prospective studies, yielding qualitatively similar results to those observed in the new data. Moreover, to help identify independent associations, we adjusted for several conventional and emerging risk factors for cardiovascular disease, including all the inflammatory cytokines studied (although we acknowledge the possibility of 'over-adjustment').
Our study also had potential limitations. First, because we used stored samples, we cannot rule out protein degradation between sample collection and assay, especially for self-activable enzymes such as MMP-9. However, randomization of samples from CHD cases and non-cases within assay plates and blinding of laboratory technicians to case–control status of the samples should have ensured that samples were treated alike during sample handling and assay. Moreover independent studies employing accelerated stability testing protocols have concluded that serum samples for determination of cytokines such as IL-6 and sIL-6R can be stored at −20°C or less for several years without affecting recovery rates, although such evidence was lacking for serum levels of the other cytokines we studied. Furthermore, despite known diurnal variation in the concentrations of some of the cytokines studied, we believe that this would also have similarly affected both CHD cases and non-cases. Second, our principal analysis used a single baseline measurement of each cytokine to study its association with incident CHD. However, our reproducibility substudies of cytokine measurements made in the same individuals an average of 12 years apart in the Reykjavik cohort and 4 years apart in the British Regional Heart Study suggest that we could have substantially underestimated any underlying aetiological associations in CHD. Conversely, the poor reproducibility of some cytokines may limit their utility for use in clinical practice as reliable risk indicators based on just a single measurement. Third, information on some extensively studied downstream measures of inflammation (such as CRP) was only available in half of the participants in our primary study (due to exhaustion of serum samples), which limited the power for direct comparisons with other cytokines, besides inability to adequately adjust for the impact of differential measurement errors in different cytokines when making such aetiological comparisons. Fourth, atherosclerosis and plaque rupture are complex processes involving the interplay between several inflammatory substances at different stages of their evolution, hence the associations may somewhat differ according to the nature of the endpoints studied. To enhance power, we used a composite coronary endpoint as our primary outcome, but subanalyses focusing solely on non-fatal MI or CHD death yielded very similar findings to those overall. Owing to limited information, we could not assess the extent to which the duration of sample storage and differences in assay reproducibility may have influenced our meta-analysis findings; nevertheless, there was little variation seen in the few reported estimates of assay reproducibility. Finally, future observational analyses will wish to evaluate these cytokines in much larger prospective studies with extensive concomitant genetic data to enable causal evaluation (e.g. Mendelian randomization).
In conclusion, several different pro-inflammatory cytokines are each associated with CHD risk independent of conventional risk factors and in an approximately log-linear manner. Although the current findings lend further support to the inflammation hypothesis in vascular disease, causality remains to be established for these cytokines in CHD.
Discussion
The current results indicate that circulating levels of several different pro-inflammatory cytokines in initially healthy people are associated with risk of CHD outcomes in an approximately log-linear manner. These associations appear to be largely independent of several conventional and emerging cardiovascular risk factors. Furthermore, updated meta-analyses reinforce the validity and generalizability of these new data, suggesting that a 1-SD higher baseline level for each of IL-6, IL-18, and TNF-α is associated with ~10–25% higher risk of non-fatal MI or CHD death. Analyses that make allowances for the considerable fluctuations that we observed of these analytes within individuals over a few years would increase the estimated size of these associations with CHD (see below). Similar associations have previously been reported for CRP and fibrinogen based on meta-analysis of individual participant data, hence we only analysed the limited data available on CRP in our primary study for making direct internal comparisons. Collectively, the current study establishes robust observational associations of several pro-inflammatory cytokines with the risk of CHD, which lends further support to the inflammation hypothesis in vascular disease. Although this does not establish causality in CHD for any of the analytes, the comprehensive evaluation of their associations with CHD should be important, given the evolving literature on cytokines as potential drug targets and gaps in translating basic scientific findings into clinical practice.
Interleukin-6 is involved in the systemic inflammatory response, but also engages in local tissue inflammation and promotes differentiation of naive T-helper cells into Th17 cells, a cell type that has been implicated in auto-immune conditions such as rheumatoid arthritis. Tumour necrosis factor-α is a pro-inflammatory cytokine, which, similar to IL-6, has been implicated in auto-immune diseases. Both IL-6 and TNF-α signalling pathways are targets of drugs (such as tocilizumab and etanercept) which are used to treat auto-immune diseases. Moreover, there have been suggestions of an increased incidence of CVD in patients with rheumatoid arthritis, and non-randomized data from biological registries suggest TNF-α blocking therapy could reduce CVD risk in these patients, although such data have well-recognized limitations. It is of interest, therefore, that two of the agents (i.e. canakinumab, a monoclonal antibody to IL-1β, and low-dose methotrexate) being tested in phase 3 trials of CVD prevention lower circulating IL-6 levels, while low-dose methotrexate therapy also lowers TNF-α levels. Furthermore, consistent with previous studies, we found circulating IL-18 levels to be positively associated with CHD incidence. In contrast, we found no significant associations of sCD40L and MMP-9 levels with CHD in the aggregate of available prospective studies, suggesting that positive findings in previous non-prospective data may have been liable to potential biases, e.g. 'reverse causality'.
Our study had several strengths. First, whereas most previous studies have concurrently considered only a few cytokines, we were able to measure simultaneously multiple pro-inflammatory cytokines in a common subset of participants in one central laboratory, facilitating uniform comparison of the observed associations across markers. Furthermore, we excluded all individuals having baseline CVD to minimize any reverse association bias. Additionally, we combined data from previously published prospective studies, yielding qualitatively similar results to those observed in the new data. Moreover, to help identify independent associations, we adjusted for several conventional and emerging risk factors for cardiovascular disease, including all the inflammatory cytokines studied (although we acknowledge the possibility of 'over-adjustment').
Our study also had potential limitations. First, because we used stored samples, we cannot rule out protein degradation between sample collection and assay, especially for self-activable enzymes such as MMP-9. However, randomization of samples from CHD cases and non-cases within assay plates and blinding of laboratory technicians to case–control status of the samples should have ensured that samples were treated alike during sample handling and assay. Moreover independent studies employing accelerated stability testing protocols have concluded that serum samples for determination of cytokines such as IL-6 and sIL-6R can be stored at −20°C or less for several years without affecting recovery rates, although such evidence was lacking for serum levels of the other cytokines we studied. Furthermore, despite known diurnal variation in the concentrations of some of the cytokines studied, we believe that this would also have similarly affected both CHD cases and non-cases. Second, our principal analysis used a single baseline measurement of each cytokine to study its association with incident CHD. However, our reproducibility substudies of cytokine measurements made in the same individuals an average of 12 years apart in the Reykjavik cohort and 4 years apart in the British Regional Heart Study suggest that we could have substantially underestimated any underlying aetiological associations in CHD. Conversely, the poor reproducibility of some cytokines may limit their utility for use in clinical practice as reliable risk indicators based on just a single measurement. Third, information on some extensively studied downstream measures of inflammation (such as CRP) was only available in half of the participants in our primary study (due to exhaustion of serum samples), which limited the power for direct comparisons with other cytokines, besides inability to adequately adjust for the impact of differential measurement errors in different cytokines when making such aetiological comparisons. Fourth, atherosclerosis and plaque rupture are complex processes involving the interplay between several inflammatory substances at different stages of their evolution, hence the associations may somewhat differ according to the nature of the endpoints studied. To enhance power, we used a composite coronary endpoint as our primary outcome, but subanalyses focusing solely on non-fatal MI or CHD death yielded very similar findings to those overall. Owing to limited information, we could not assess the extent to which the duration of sample storage and differences in assay reproducibility may have influenced our meta-analysis findings; nevertheless, there was little variation seen in the few reported estimates of assay reproducibility. Finally, future observational analyses will wish to evaluate these cytokines in much larger prospective studies with extensive concomitant genetic data to enable causal evaluation (e.g. Mendelian randomization).
In conclusion, several different pro-inflammatory cytokines are each associated with CHD risk independent of conventional risk factors and in an approximately log-linear manner. Although the current findings lend further support to the inflammation hypothesis in vascular disease, causality remains to be established for these cytokines in CHD.
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