Our results support three previous studies that observed higher levels of the pro-inflammatory cytokine TNFα among post-menopausal women [31
], although others did not observe such a difference [34
], or observed lower levels [36
]. Two soluble cytokine receptors, sIL-1RII and sIL-2Ra were also higher among post- vs. pre-menopausal women. The associations were apparent after adjustment for age and BMI, which suggests that these markers may be influenced by sex hormones.
Increasing parity was associated with higher levels IL-12p40, although this association was no longer statistically significant after adjustment for smoking. We are unaware of previous reports that have evaluated the association between previous pregnancies and inflammation markers. However, a microarray study of normal breast tissue found that several inflammation-associated genes were upregulated in both recently (0–2 years since pregnancy) and distantly (5–10 years since pregnancy) parous vs. nulliparous women [37
]. However, despite adjustment for age and BMI, we cannot exclude the possibility that the association may have been confounded by other factors, including age at each pregnancy and time since last pregnancy.
Women with a first degree family history of breast or ovarian cancer had lower levels of several cytokines. While inflammation marker levels are not likely to be directly related to family history, this risk factor may be associated with adoption of healthy lifestyle elements.
Current use of NSAIDs was inversely associated with a number of inflammatory markers (IL-1β, IL-2, IL-10 and IL-12p70), in line with our expectations. However, NSAID use was not associated with levels of CRP. Prior reports on the association between NSAID use and CRP are conflicting [38
], which may be due to differences in duration, types, and doses of NSAID evaluated, but also because some studies were limited in sample size [38
], included subjects with existing chronic disease [43
], or reported associations for regular users who were asked to abstain from use prior to blood sampling [41
Somewhat contrary to our expectations, smoking status was not significantly associated with CRP, IL-6, TNFα or other pro-inflammatory cytokines. In our study, current smokers generally had lower
levels of cytokines than non-smokers, though these associations were only statistically significant for the anti-inflammatory markers, IL-4, IL-13 and IL-12p40. Lower levels of these cytokines among smokers vs. non-smokers were observed in all four sub-studies (NYUWHS-OVCA, NYUWHS-NHL, ORDET, and NSHDS). Previous studies on current smoking and cytokines among healthy individuals have found positive [34
], inverse [51
], and null [49
] associations. Inconsistent findings may be a result of incomplete control for lifetime exposure to cigarette smoke (e.g. pack-years), which may be a more relevant measure of exposure than current smoking status.
CRP was not correlated with most cytokines. Given the role of IL-6, and to a lesser extent TNFα and IL-1β, in the induction of CRP, we expected to observe moderate to strong correlations of CRP with these cytokines. In models adjusted for study and age (but not BMI), CRP was positively correlated with TNFα (r = 0.1, P <
0.005) and IL-6 (r= 0.1, P=0.001), but not IL-1β. Others have found a moderate correlation between CRP and IL-6, some of which adjusted for BMI, with most estimating a correlation coefficient between 0.3–0.5 [2
]. However, data from the Women’s Health Initiative trial suggests that there could be IL-6-independent pathways for regulating CRP, as CRP and IL-6 had different associations with several cardiovascular risk factors: HRT use, alcohol use, and exercise [44
]. For example, the consistent association between CRP and HRT [58
] vs. the weak or null association between IL-6 and HRT, may be due to direct (IL-6-independent) hepatic induction of CRP [44
The observed significant correlation between TNFα, IL-6, and IL-1β was expected due to the regulatory inter-relationship of these cytokines [34
]. Cytokines were correlated with each of the other cytokines, and less so with cytokine modulators. In physiological conditions, elevations in pro-inflammatory cytokines trigger elevations in anti-inflammatory cytokines to resolve the inflammatory response. Thus, we expected all pro- and anti-inflammatory cytokines to be positively correlated with each other in healthy women. However, since all the cytokines were measured using the same assay kit, we cannot rule out the possibility that imperfect antibody specificity could have contributed to the strength of the correlations. The relationship between cytokines and their modulators is complex, because soluble cytokine receptors can act as both cytokine agonists and/or antagonists [61
]. Possible reasons for the lack of correlation between cytokines and their modulators have been suggested by others, such as the longer half-life of soluble receptors in circulation and/or the inability to detect all unbound and bound forms of cytokines with immunoassays [62
The lack of association between cytokines and 25(OH)D, the best representation of an individual’s vitamin D status, is in agreement with a study of vitamin D supplementation in overweight and obese individuals that did not find an association between circulating 25(OH)D and CRP or cytokines (including IL-2, IL-4, IL-5, IL-10, IL-12, IL-13) [52
]. We found some evidence of a positive association for IL-13 and 25(OH)D, though the association was not significant in multivariate analyses. Others have reported a lack of association between 25(OH)D and several inflammation markers (CRP, IL-1β, IL-6, IL-10, SIL-2R, TNFα, sTNF-R1, and sTNF-R2) [65
], though one small study found an inverse association between 25(OH)D and TNFα [65
] and two others found that vitamin D supplementation stabilized [71
] or reduced [72
] TNFα among patients with congestive heart failure or obesity, respectively. We note that the median 25(OH)D level in our study group (median: 48 nmoI/L, interquartile range: 25, 81 nmoI/L) is considered to be in the “insufficient” range (~<50 nmoI/L) based on some recommendations [73
This study has a number of strengths. First, participants were healthy and not using HRT or OCs for at least 6 months prior to blood sampling, thus minimizing the effects of existing disease and exogenous hormones on cytokine levels. Second, we evaluated associations between risk factors and a large number of cytokines and cytokine modulators which have not been evaluated in previous studies. Third, all inflammation marker measurements were performed in the same laboratory, which minimizes assay variability and allows individuals to be ranked relative to others.
Our study also has some limitations. First, questionnaires differed between cohorts. However, results were usually consistent across cohorts, providing confidence in our findings. Second, it is possible that measured levels of a single cytokine are not reflective of their biologically relevant concentration. This may be due to assay limitations in quantification of absolute levels and/or the presence of circulating cytokine inhibitors, including soluble receptors and other binding proteins. Finally, the exploratory nature of our analyses resulted in a substantial number of statistical tests, thus it is likely that some associations were significant due to chance. However, we did not adjust for multiple comparisons because our goal was to identify general patterns of association that may explain risk factor associations and to aid in the selection of potential confounders for future studies of cytokines and chronic disease risk. The associations observed here should be evaluated in independent studies.
In conclusion, we observed the expected relationships between inflammation markers and age and BMI. Higher levels of certain markers were observed among postmenopausal vs. premenopausal women (TNFα, sIL-1RII, and sIL-2Ra) and with increasing parity (IL-12p40). Lower levels were observed among current versus non-users of NSAIDs (IL-10, IL-β, IL-10, IL-12p70, and IL-12p40), women with a family history of breast or ovarian cancer (IL-4, IL-10, and IL-13), and current versus non-smokers (IL-4, IL-13, and IL-12p40). These findings suggest that one mechanism underlying the relationship between reproductive and lifestyle factors and chronic diseases may involve inflammation mediators.