In this study, we compared the monocyte glucocorticoid sensitivity between middle aged men and women while controlling for known risk factors for cardiovascular disease. We used an in vitro assay to measure monocyte cytokine release in response to a standardised dose of lipopolysaccharide and to assess the extent to which this cytokine release was inhibited by increasing concentrations of two glucocorticoids.
The main finding of our study is that, even when controlling for well known cardiovascular risk factors, blood samples from men required larger quantities of either dexamethasone or hydrocortisone to inhibit lipopolysaccharide stimulated release of IL-6 and TNFα than did samples from women, while the cytokine release without glucocorticoid co-incubation was higher in samples from men than in those from women. The findings suggest higher proinflammatory activity in blood monocytes of men than in monocytes harvested from women. This notion becomes even stronger given that we controlled for established cardiovascular risk factors. Previous studies have shown that, on average, men have higher body mass index, higher LDL:HDL ratio, and higher systolic and diastolic blood pressures than women.1,4,34,35
Therefore, to prevent confounding, cardiovascular risk factors need to be controlled for when testing for a unique sex difference in monocyte glucocorticoid sensitivity.
What may the clinical implications of our findings be and how do they fit with the literature? Our findings indicate that the proinflammatory response of monocytes to an equal amount of lipopolysaccharide, such as that produced by bacteria present within an atherosclerotic lesion, is higher in men than in women. Corroborating these findings, some investigators found greater TNFα release with lipopolysaccharide stimulation in men than in women36,37
and with IL-6,37
while others did not detect such differences.14,38
Further driving such controversy, Rohleder and colleagues14
found lower sensitivity of monocyte IL-6 release with dexamethasone inhibition in men than in women, while Daun and colleagues39
did not find such a sex difference in hydrocortisone regulation of lipopolysaccharide stimulated production of IL-1β from peripheral blood mononuclear cells. In contrast to our study, however, those studies had relatively smaller population samples that consisted mainly of young and healthy people who conceivably have a lower cardiovascular risk than our middle aged participants.
Epidemiological data support the notion that increased plasma concentrations of TNFα and IL-6 increase the risk of coronary artery disease.10–12,40
Thus, differential regulation of monocyte TNFα and IL-6 production may provide a link between sex and the progression of atherosclerotic lesions. Our data suggest that monocytes from men not only are predisposed towards more pronounced release of TNFα and IL-6 but also are less sensitive to endogenous signals (glucocorticoids) responsible for curtailing inflammatory responses. This may lead to a more rapid development of atherosclerosis in men than in women.1
Although speculative, one possible explanation for observed sex differences in lipopolysaccharide stimulated cytokine release and in glucocorticoid sensitivity may relate to different concentrations of gonadal steroids. The female immune system is influenced by both oestrogens and progesterones. Oestrogens may dose dependently affect lipopolysaccharide associated changes in immune responses of macrophages.13,41
Compared with men, women in the luteal phase of the menstrual cycle had diminished monocyte release of TNFα and IL-6 with lipopolysaccharide.37
In that study, these effects correlated with the concentration of oestradiol in plasma. The male immune system, on the other hand, is under the control of testosterone and other androgens, which apparently do not affect macrophage function.42–45
Despite increasing knowledge of the pathways through which gonadal steroids and glucocorticoids influence immunological target tissues, it remains unknown how these steroids interact at peripheral inflammatory sites. The activated oestrogen receptor has been shown to inhibit the transcription factors nuclear factor κB and activator protein 1.46,47
Progesterone, because of its structural similarities with the glucocorticoids receptor, has been reported to increase cytosolic mRNA concentrations of the nuclear factor κB inhibitor.48
By these mechanisms, both female gonadal steroids seem to exert anti-inflammatory effects.
Da Silva has proposed several mechanisms by which gonadal steroids may modulate the sensitivity of target tissues towards glucocorticoids.49
In animal and cell culture studies, 17β oestradiol has been show to decrease the expression of the glucocorticoid receptor and thereby to decrease glucocorticoid sensitivity.50
Furthermore, expression of heat shock proteins 70 and 90, which are essential in keeping the glucocorticoid receptor in the cytoplasm in an inactive state (although it can be ligand activated), are modulated by female gonadal steroids.51,52
Several limitations of our study require consideration. Firstly, our investigation focused on a working population and, thus, may not be generalisable to clinical populations and to people with overt atherosclerotic disease in particular. Secondly, we can only speculate as to whether the observed differences between men and women are clinically relevant. Thirdly, as we did not assess sex steroids, possible explanations for the observed sex differences in cytokine release and glucocorticoid sensitivity are not unequivocally attributable to hormonal issues. Fourthly, we compared very different group sizes of men and women potentially resulting in different variances of mean values of variables of interest. While Levene’s test showed that variances of uninhibited lipopolysaccharide stimulation of TNFα and IL-6 and of the four IC50 estimates were equal, three of the 20 computations across the four inhibition curves suggested a violation of this assumption (data not shown). Nonetheless, since we built our argument on sex differences in uninhibited stimulation of proinflammatory cytokines and calculated IC50 values, we feel that this caveat did not invalidate our findings.
In summary, our data show that monocytes from middle aged men have reduced responsiveness to glucocorticoids even when established cardiovascular risk factors are controlled for. This finding of reduced glucocorticoid sensitivity in men implies relatively sustained cytokine production once monocytes have encountered stressful stimuli (such as lipopolysaccharide). Such a mechanism may be one possible biological pathway linking sex to progression of atherosclerotic disease and increased coronary risk in men.