The present study provides further evidence that brief laboratory stress primes peripheral immune cells to produce higher levels of pro-inflammatory cytokines in response to endotoxin, here among a community sample of relatively healthy midlife men and women. Consistent with a growing literature suggesting that acute stress is associated with activation of innate inflammatory pathways (Steptoe et al., 2007
), we found that a public speaking task resulted in significant increases in cellular production of IL-6, TNF-α, and to a lesser extent, IL-1β, as measured 30 minutes after task completion. Although there were no gender differences in the magnitude of the increase in cytokine production from baseline to 30 minutes post-task, we did observe differences in stimulated production of IL-6, TNF-α, and IL-1β from baseline to immediately post-task, with men showing a significant decrease and women no change. Thus, there were notable gender differences in the pattern of cytokine production responses to acute stress, with women showing no change from baseline to post-task, but a significant increase during the 30 minute recovery period and men showing a more biphasic response, with a significant decrease from baseline to post-task and an increase during recovery. Interestingly, this pattern of gender differences is inconsistent with the only other study that we know of that has examined gender differences in cytokine production following acute stress. In this study, Rohleder (2001)
showed a significant decrease in stimulated production of IL-6 from before to 60 minutes after the Trier Social Stress Test for males, but no change for females. One possible explanation for the inconsistent findings across these studies is differences in the timing of measures, with stress-related increases in cytokine production having returned to baseline levels by 60 minutes post-task. In this regard, evidence suggests that many immune responses to acute stress return to baseline levels precipitously following the end of the stressor (Segerstrom and Miller, 2004
). Thus, further research is warranted to better delineate the role gender plays in modulating inflammatory response trajectories following acute challenge.
The speech stressor used in the current study was associated with significant increases in HR and BP, yet we did not observe gender differences in the magnitude of these effects. This raises questions regarding the mechanism of gender differences in cytokine production in response to the task. Convincing animal and in vitro evidence suggests that the autonomic nervous system can up- and down-regulate production of pro-inflammatory cytokines (Nance and Sanders, 2007
). For example, experimental evidence shows that stress-induced increases in catecholamines upregulate the NF-kB signaling pathways responsible for pro-inflammatory cytokine production (Bierhaus et al., 2003
). Conversely, LPS-stimulated cells treated with catecholamines or β-agonists show decreased pro-inflammatory cytokine production (Izeboud et al., 1999
; van der Poll et al., 1994
). Recent evidence also suggests that the parasympathetic division of the ANS can regulate inflammatory competence in real time via the vagus nerve (Pavlov and Tracey, 2005
). Indeed, cross-sectional evidence shows that heart rate variability, an index of sympathovagal balance, covaries inversely with stimulated pro-inflammatory cytokine production (Marsland et al., 2007a
; Sloan et al., 2007
), and that this relationship appears to be moderated by gender (O’Connor et al., 2007
). This raises the possibility that gender differences in sympathovagal activation in response to acute stress may contribute to differences in the inflammatory response.
Our findings suggest that menopausal status partially accounts for gender differences in stress-induced cytokine production, with post-menopausal women showing greater increases in cytokine production from baseline to immediately following the stressor than pre-menopausal women or men. Though preliminary, given small sample sizes, this is the first study to document menopausal differences in stress-induced cytokine reactivity. Hormonal differences between pre and postmenopausal women may help explain these findings. Plasma concentrations of estradiol are lower in postmenopausal women (< 15 pg/ml) than premenopausal women (50–250 pg/ml) or men (50 pg/ml) (Walsh and Shiff, 2001
; Winters, 2001
). Estradiol acts to inhibit pro-inflammatory cytokine gene expression, NF-kB binding, and production of pro-inflammatory cytokines (Deshpande et al., 1997
; Liu et al., 2005
; Ray et al., 1997
). Thus, it is possible that the increased inflammatory reactivity we observed among post-menopausal women is related to lower circulating levels of reproductive hormones. Further research on the effects of stress on cytokine production in immune cells treated in vitro with reproductive hormones is warranted. Moreover, future studies are needed to explore whether this increased inflammatory propensity contributes to the heightened susceptibility to inflammatory disease that is observed among older women.
The present findings should be interpreted in context of a number of study limitations. First, this study did not include a control condition that would allow us to rule out alternative explanations for stress-related changes in cytokine production, such as prolonged IV catheterization and repeated blood draws. Levels of circulating pro-inflammatory mediators are largely unaffected by these factors (Steptoe et al., 2001
; von Kanel et al., 2006
); however, it remains unclear whether this is the case for the cellular production of cytokines (Steptoe et al., 2007
). Second, information regarding menstrual phase was not collected in our sample. Given that reproductive hormones, including progesterone and estrogen, regulate inflammatory activity, future work should consider menstrual phase in interpreting gender and menopausal status effects. In this regard, Rohleder and colleagues (2003)
reported a modest increase in IL-6 production following a laboratory stressor among women taking oral contraceptives whereas levels among women in their luteal phase decreased. In the current study, only one premenopausal woman reported taking oral contraceptives; however, the reported findings remained unchanged when this person was dropped from the analyses. Finally, it should be noted that demonstrating gender differences in the magnitude of responses to a speech task offers only limited evidence for gender differences in stress-induced cytokine reactivity. To conclude that there are generalized gender differences will require that the current findings be replicated and reproduced with diverse behavioral stressors.
The current study employed a whole blood assay designed to permit the quantification of cytokines produced by the full array of immune cells acting in concert within their normal milieu and is thought to simulate the in vivo environment (De Groote et al., 1992
). In contrast, others have measured cytokine production by activated monocytes or mononuclear cells (e.g., Suarez et al., 2003
), which provides greater specificity, but a less accurate picture of the total immune response. Evidence suggests that these differing methods can influence results. For example, in samples taken from the same healthy individuals, the stimulation of isolated mononuclear cells resulted in higher levels of IL-1 than the stimulation of whole blood cultures that contained identical concentrations of mononuclear cells, suggesting a downregulation of the cytokine response by elements in whole blood (DeGroote et al., 1992
). Given the goals of the current study, we selected to use a whole blood assay that offers a closer approximation of in vivo immune processes. Future work may benefit from the isolation of cells to permit an examination of the specific immune cells contributing to the observed inflammatory response (e.g. intracellular cytokine staining for flow cytometry).
It is well documented that women are more susceptible than men to a number of inflammatory conditions (e.g., autoimmune diseases) (Gleicher and Barad, 2007
; Lockshin, 2001
). However, the mechanisms of these differences are poorly elucidated. Although gender differences in cytokine production may contribute to this differential risk, it is important to note that the clinical significance of individual differences in pro-inflammatory cytokine production remain unclear. That said, initial evidence shows a positive association of stimulated cytokine production with health risk. For example, higher levels of LPS-stimulated inflammatory activity have been observed in patients with rheumatoid arthritis relative to healthy controls (Scuderi et al., 2003
) and among post myocardial infarction patients who go on to experience heart failure when compared with those who do not (Satoh et al., 2006
). Thus, further prospective investigation of gender differences in stress induced cytokine reactivity is justified to determine whether individual response patterns contribute to susceptibility to inflammatory disease.