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Estrogen Modulates the Hypothalamic-Pituitary-Adrenal and Inflammatory Cytokine Responses to Endotoxin in Women¹

Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032

Address all correspondence and requests for reprints to: Dr. Sharon L. Wardlaw, Department of Medicine, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. E-mail: sw22{at}columbia.edu

Abstract

Endotoxin stimulates the release of the inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-, which are potent activators of the hypothalamic-pituitary-adrenal (HPA) axis. Recent studies in the rodent and in the primate have shown that the HPA responses to endotoxin and IL-1 were enhanced by gonadectomy and attenuated by estradiol (E2) replacement. In addition, there is some evidence, in the rodent, that estrogen modulates inflammatory cytokine responses to endotoxin. To determine whether estrogen has similar effects in humans, we studied the cytokine and HPA responses to a low dose of endotoxin (2–3 ng/kg) in six postmenopausal women with and without transdermal E2 (0.1 mg) replacement. Mean E2 levels were 7.3 ± 0.8 pg/mL in the unreplaced subjects and increased to 102 ± 13 pg/mL after estrogen replacement. Blood was sampled every 20 min for 1–2 h before, and for 7 h after, iv endotoxin administration. Endotoxin stimulated ACTH, cortisol, and cytokine release in women with and without E2 replacement. E2 significantly attenuated the release of ACTH (P < 0.0001) and of cortisol (P = 0.02). Mean ACTH levels peaked at 190 ± 91 pg/mL in the E2-replaced group vs. 411 ± 144 pg/mL in the unreplaced women, whereas the corresponding mean cortisol levels peaked at 27 ± 2.9 µg/dL with E2 vs. 31 ± 3.2 µg/dL without E2. Estrogen also attenuated the endotoxin-induced release of IL-6 (P = 0.02), IL-1 receptor antagonist (P = 0.003), and TNF- (P = 0.04). Mean cytokine levels with and without E2 replacement peaked at 341 ± 94 pg/mL vs. 936 ± 620 pg/mL for IL-6, 82 ± 14 ng/mL vs. 133 ± 24 ng/mL for IL-1 receptor antagonist, and 77 ± 46 pg/mL vs. 214 ± 87 pg/mL for TNF-, respectively. We conclude that inflammatory cytokine and HPA responses to a low dose of endotoxin are attenuated in postmenopausal women receiving E2 replacement. These data show, for the first time in the human, that a physiological dose of estrogen can restrain cytokine and neuroendocrine responses to an inflammatory challenge.

BACTERIAL ENDOTOXINS, lipopolysaccharides present in the cell wall of gram-negative bacteria, induce a variety of host acute phase responses and have been widely used to stimulate endogenous cytokine release and to mimic some of the events that occur during sepsis (1, 2). Endotoxin stimulates the synthesis and release of inflammatory cytokines from peripheral monocytes and macrophages and activates the hypothalamic-pituitary-adrenal (HPA) axis. The stimulation of the HPA axis by endotoxin is mediated primarily through the release of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)- (3). All three cytokines have been shown to independently activate the HPA axis. They act predominantly at the level of the hypothalamus and exert synergistic effects, with respect to stimulation of the HPA axis (3). Recent studies in the rodent and in the primate have shown that gonadal steroids can modulate the response of the HPA axis to inflammatory stimuli. In the rodent, the HPA responses to endotoxin and to IL-1 were enhanced by gonadectomy and attenuated by estradiol (E2) and testosterone replacement (4, 5, 6). Studies in ovariectomized monkeys have also shown that the HPA response to IL-1 is attenuated by E2 replacement (7). The mechanisms underlying these observations are unclear but may involve gonadal steroid modulation of cytokine and neuropeptide responses. For example, several studies have documented an inhibitory effect of E2 on IL-6 gene expression (8, 9). There are, however, no studies examining the effects of estrogen on circulating levels of inflammatory cytokines, or on the HPA axis, in response to endotoxin in humans. We have therefore studied the effects of a replacement dose of E2 on the inflammatory cytokine and HPA responses to a low dose of endotoxin in postmenopausal women.

Materials and Methods

Experimental protocol

Six healthy female subjects, 42–68 yr old, were studied. Four subjects were at least 1 yr after natural menopause, and two had undergone a bilateral oophorectomy, 4 and 15 yr before. The subjects were taking no medications other than vitamin or mineral supplements. Specifically, there was no use of oral or inhaled steroids. No aspirin or nonsteroidal antiinflammatory drug was taken for at least 48 h before the study. Subjects received low-dose endotoxin on two occasions separated by 1–2 months. Subjects were studied in random order, in a nonblind fashion, either with or without estrogen replacement. The mean (±SEM) concentration of E2 in peripheral blood was 7.3 ± 0.8 pg/mL before the study in the unreplaced subjects. Low E2 levels were documented for at least 1 month before the study. Each subject was treated with a transdermal E2 patch (0.1 mg) for 1 month. E2 levels were measured 4 days before the study in most women; and, if the level was less than 50 pg/mL, an additional E2 patch was added. On the day of the study, the mean E2 concentration was 102 ± 14 pg/mL. Individual E2 concentrations are shown in Table 1. An indwelling iv catheter was inserted between 0800 h and 0900 h on the day of the study. Purified endotoxin was given iv at 1000 h, and blood was sampled every 20 min for 1–2 h before, and for 7 h after, endotoxin injection. Blood samples were centrifuged within 1 h, and plasma was separated and stored at -20 C for cytokine and hormonal assays.

Informed consent was obtained from all subjects, and the study was approved by the Columbia-Presbyterian Medical Center Institutional Review Board.

Hormone and cytokine assays

ACTH was assayed by RIA in extracted plasma in the first four subjects and by immunoradiometric assay (IRMA) in unextracted plasma in the last two subjects. ACTH RIA was performed with an antiserum directed against ACTH(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) (IgG Corporation, Nashville, TN) with an assay sensitivity of 5 pg/mL (10). The IRMA (Nichols Institute Diagnostics, San Juan Capistrano, CA) has a detection limit of 5 pg/mL. Neither the ACTH RIA nor IRMA cross-reacts with -melanocyte-stimulating hormone, corticotropin-like intermediate lobe peptide, CLIP, or ß-endorphin. Serum cortisol was assayed in unextracted plasma by solid-phase RIA (Diagnostic Products, Los Angeles, CA). Two assays were used for E2 measurements: All samples were measured by a commercial solid-phase, chemiluminescent immunoassay (Immulite, Diagnostic Products) with an assay sensitivity of 20 pg/mL. In samples with E2 levels of less than 20 pg/mL, the measurement was repeated using a sensitive double-antibody RIA for E2 (Diagnostic Products). Sensitivity of this assay was 5 pg/mL. All cytokines were assayed by specific monoclonal human sandwich immunoassays with solid-phase enzyme-linked immunosorbent assay kits. Their lower limit of sensitivity varied, depending on the dilutions measured, but each subject had the same dilution performed in both parts of the study. The detection limit for IL-6 and IL-1 receptor antagonist (IL-1 ra) (both R&D Systems, Minneapolis, MN) ranged between 3.1–12.5 pg/mL and 47–188 pg/mL, respectively. The detection limit for TNF- (R&D Systems and Genzyme Diagnostics, Cambridge, MA) ranged between 20–31 pg/mL.

E2 levels were measured twice at the start of each study, before endotoxin injection. Cytokines and cortisol were measured every 20–60 min, whereas the interval for ACTH measurement was 20 min.

Data analysis

The effects of endotoxin on hormone and cytokine responses in both groups were analyzed by ANOVA with repeated measures. Statistical comparisons between +E2 and -E2 groups, over time, were performed using Bonferroni-Dunn post hoc analysis. Total area under the hormone and cytokine response curves (AUC) was calculated by trapezoid analysis, and the responses in both groups were compared by paired Wilcoxon signed-rank test. Individual peak hormone and cytokine levels in both groups were compared by paired Wilcoxon signed-rank test.

Results

ACTH and cortisol responses

The ACTH and cortisol responses to iv endotoxin are shown in Fig. 1. There was a significant stimulatory effect of endotoxin on ACTH and cortisol over time, both without and with E2 replacement (P < 0.0001). Mean (±SEM) ACTH levels, measured during the baseline hour, were 8.7 ± 1.3 pg/mL with E2 vs. 12.9 ± 1.4 pg/mL without E2 treatment, and levels rose to a peak of 190 ± 91 pg/mL with E2 vs. 411 ± 144 pg/mL without E2. Cortisol increased from a mean baseline of 7.7 ± 1.2 µg/dL with E2 vs. 7.7 ± 0.7 µg/dL without E2 replacement and peaked at 27.4 ± 2.9 µg/dL with E2 vs. 30.8 ± 3.2 µg/dL without E2. Whereas there was no significant effect of E2 on baseline levels of ACTH or cortisol, E2 significantly attenuated the release of both ACTH (P < 0.0001) and of cortisol (P = 0.02) in response to endotoxin. The AUC calculated for ACTH during the 1- to 6-h period after endotoxin administration in the E2-replaced women was 54.6% of the AUC in the unreplaced subjects (P = 0.03). The AUC calculated for cortisol after endotoxin in the E2-replaced women was 84.8% of the AUC in the unreplaced subjects (P = 0.12). Individual peak ACTH and cortisol responses are shown for each subject in Table 1.

View larger version (18K): [in this window] [in a new window]   Figure 1. Mean (±SEM) plasma ACTH (upper panel) and cortisol (lower panel) responses to endotoxin in six postmenopausal women, studied twice, without (-E2) and with (+E2) E2 replacement. There was a significant stimulatory effect of endotoxin on ACTH and cortisol, over time, both without and with E2 replacement (P < 0.0001). E2 significantly attenuated the release of ACTH (P < 0.0001) and of cortisol (P = 0.02) over time.

The effects of endotoxin on IL-6, IL-1ra, and TNF- release into peripheral blood are shown in Figs. 2, 3, and 4. A significant stimulatory effect of endotoxin on IL-6 release (P < 0.01) and IL-1ra release (P < 0.0001), over time, was noted in the presence and absence of E2. A significant stimulatory effect of endotoxin on TNF- release (P < 0.01) was noted only in the absence of E2. Baseline values for IL-6 were below the level of assay detection, both with and without E2; mean IL-6 levels peaked at 341 ± 94 pg/mL with E2 replacement, compared with 936 ± 620 pg/mL without E2. Baseline levels of IL-1ra were 0.13 ± 0.03 ng/mL with E2 vs. 0.20 ± 0.06 ng/mL without E2 and peaked at 82 ± 14 ng/mL and at 133 ± 24 ng/mL, with and without E2, respectively. Baseline TNF- levels were below the level of assay detection, both with and without E2 treatment and increased to 77 ± 46 pg/mL and 214 ± 87 pg/mL, respectively, after endotoxin. Estrogen significantly attenuated the release of IL-6 (P = 0.02), IL-1ra (P = 0.003), and TNF- (P = 0.04). The AUC calculated for IL-6 during the 1- to 6-h period, after endotoxin injection in the group on E2, was 31.3% of the AUC in the unreplaced group (P = 0.03). The AUC calculated for IL-1ra, 2–7 h after endotoxin administration in the E2-replaced women, was 61.75% of the AUC without E2 (P = 0.04). The AUC for TNF-, 0–3 h after endotoxin administration in the E2-treated women, was 49% of the AUC in the unreplaced group (P = 0.03). Individual peak cytokine responses are shown for each subject in Table 1.

View larger version (13K): [in this window] [in a new window]   Figure 2. Mean (±SEM) plasma IL-6 responses to endotoxin in six postmenopausal women, studied twice, without (-E2) and with (+E2) E2 replacement. Endotoxin stimulated the release of IL-6, over time, in both groups (P < 0.01). E2 attenuated the endotoxin-induced release of IL-6 over time (P = 0.02).

View larger version (13K): [in this window] [in a new window]   Figure 3. Mean (±SEM) plasma IL-1ra responses to endotoxin in six postmenopausal women, studied twice, without (-E2) and with (+E2) E2 replacement. Endotoxin stimulated the release of IL-1ra, over time, in both groups (P < 0.0001). E2 attenuated the endotoxin-induced release of IL-1ra over time (P = 0.003).

View larger version (13K): [in this window] [in a new window]   Figure 4. Mean (±SEM) plasma TNF- responses to endotoxin in six postmenopausal women, studied twice, without (-E2) and with (+E2) E2 replacement. E2 attenuated the endotoxin-induced release of TNF- over time (P = 0.04).

The normal physiological response to inflammation is modulated by the HPA axis, which is activated by inflammatory cytokines produced during the immune response (3). Bacterial endotoxins stimulate the synthesis and release of the inflammatory cytokines IL-1, IL-6, and TNF-, which stimulate CRH and AVP in the hypothalamus, with subsequent pituitary-adrenal activation (3, 11, 12). The resulting increase in adrenal glucocorticoid secretion has well-documented inhibitory effects on the inflammatory process and on inflammatory cytokine release. Previous animal studies have demonstrated that gonadal steroids may also play a role in modulating the neuroendocrine and cytokine responses induced by inflammation. Our data now show, for the first time in humans, that estrogen attenuates the HPA response to a low dose of endotoxin in vivo. This was accompanied by a parallel decrease in the endotoxin-induced stimulation of the inflammatory cytokines IL-6 and TNF-, as well as IL-1ra, after estrogen treatment.

Previous studies in the rodent and in the nonhuman primate have shown that the HPA responses to endotoxin and IL-1 were enhanced by castration and attenuated by sex steroid replacement (4, 6, 7, 13, 14). In mice, gonadectomy did not modify basal plasma corticosterone levels, but the administration of endotoxin induced significantly higher corticosterone levels in ovariectomized animals, compared with either E2-replaced or intact female mice (4). Similarly, gonadectomy significantly augmented IL-1-induced corticosterone release in female rats (13). In ovariectomized rhesus monkeys, the cortisol response to an intracerebroventricular infusion of IL-1 was attenuated by E2 treatment, which produced plasma concentrations found in the early-midfollicular phase of the cycle (7). More recently, another study in ovariectomized monkeys showed that estrogen decreased the ACTH response to an iv injection of IL-1ß (15). Gonadal steroids have also been reported to modulate the HPA response to a variety of other types of noninflammatory stress, such as neurogenic or psychosocial stress. The HPA response to these stresses differs in male and female animals, with higher responses generally being reported in the female. The effects of sex steroids on the HPA response to these types of stress depends on the specific stress employed and on the gender of the animal (16, 17, 18). In the ovariectomized female rat, for example, the ACTH and corticosterone responses to footshock and to ether stress were enhanced by E2 treatment (16). In contrast, in the male rat, the ACTH response to restraint stress was enhanced by castration and attenuated by testosterone replacement (18). In human studies, the results have been variable with respect to the effects of sex steroids on the HPA response to psychosocial stress. E2 has been reported to both enhance and attenuate the HPA response to psychosocial stress (19, 20). In both male and female animals, however, although there are gender differences in the HPA responses to endotoxin and IL-1, these responses are enhanced by castration and attenuated by androgen and estrogen replacement (4, 13). We now show that although basal plasma ACTH and cortisol levels were not significantly different in the postmenopausal women with and without E2 replacement, both the ACTH and cortisol responses to endotoxin were blunted by E2. Thus, both the previous animal studies and our current human study all show that E2 attenuates the HPA response to endotoxin.

The mechanisms responsible for the estrogen-induced attenuation of the HPA response to endotoxin are, at present, not clear. Potential mechanisms include estrogen modulation of inflammatory cytokines, of CRH and AVP in the hypothalamus, or of other inflammatory mediators, such as the cyclooxygenase or nitric oxide (NO) pathways, which can influence hypothalamic CRH release and the subsequent pituitary-adrenal response. In the rodent, sex steroids have been shown to affect CRH and AVP gene expression in the hypothalamus (18, 21, 22, 23, 24). Other studies in the rodent show that cytokine-induced activation of the HPA axis can be blocked by inhibitors of the cyclooxygenase pathway and enhanced by inhibitors of NO synthesis, indicating a role for PGs and NO in this process (12, 25, 26). Thus, endogenous NO seems to restrain the HPA response to inflammatory stimuli. A temporal correlation has been demonstrated between endotoxin-induced activation of the HPA axis and stimulation of neuronal NO synthase in the paraventricular nucleus of the hypothalamus (27). Endotoxin has also been shown to stimulate the production of inducible NO synthase in the brain (28). Sex steroid-induced alterations in NO production have been postulated to play a role in modulating HPA responses to inflammation. E2 has been shown to increase expression of neuronal NO synthase messenger RNA (mRNA) in the rat hypothalamus (29). E2 has also been shown to increase circulating NO levels in postmenopausal women (30). It is thus possible that the estrogen-induced stimulation of NO could contribute to the suppressed HPA response to endotoxin.

In the current study, we show that the cytokine response to endotoxin is also attenuated by E2. Plasma levels of the inflammatory cytokines TNF- and IL-6 were reduced after E2 treatment, as was the level of IL-1ra, an endogenous antagonist of IL-1, which is secreted in parallel with IL-1ß. Because TNF-, IL-1ß, and IL-6 exert synergistic effects, with respect to endotoxin-induced stimulation of the HPA axis, it is possible that the attenuated cytokine responses are, at least in part, responsible for the blunted HPA response to endotoxin. IL-6 plays a major role in this process, as shown by the fact that antibodies against IL-6 almost completely block the ACTH response to endotoxin in mice (31). There is considerable evidence that estrogen has direct inhibitory effects on IL-6 synthesis and release. IL-6 secretion by bone-marrow-derived stromal cells and osteoblasts from rodents and humans is inhibited by E2, as is IL-6 gene expression (32). E2 has been reported to inhibit the human IL-6 gene through an estrogen-receptor-mediated indirect effect on IL-6 transcription (8, 9). Circulating IL-6 levels, measured with a high-sensitivity assay, have also been reported to be lower in postmenopausal women receiving hormone replacement therapy (33). In our study, we did not detect a difference in baseline plasma IL-6 levels with and without E2 treatment. However, the IL-6 assay used in our study was not as sensitive as the assay employed by Straub et al. (33). E2 has also been shown to inhibit TNF- and IL-1ß release from peripheral mononuclear cells in postmenopausal women (34, 35, 36). In addition, ovariectomy has been shown to significantly enhance endotoxin-induced TNF- release in mice (4).

Because endotoxin and IL-1ß both stimulate IL-6, we questioned, in a recent study in the monkey, whether the estrogen-induced suppression of the HPA response to endotoxin and IL-1 results directly from decreased IL-6 release. We compared the ACTH response to either IL-6 (which does not stimulate IL-1 or TNF- release) or IL-1ß in ovariectomized monkeys, with and without 3 weeks of E2 replacement. Physiological levels of E2 actually enhanced the ACTH response to IL-6 but attenuated the ACTH response to IL-1. This attenuated ACTH response to IL-1 was accompanied by suppressed IL-6 plasma levels (15). Our results in the monkey suggest that the blunted HPA response to IL-1 can be explained, at least in part, by E2-induced alterations in IL-6 release. Our current results in E2-treated women suggest that a similar mechanism may also exist in the human.

In summary, we conclude that E2 attenuates the endotoxin-induced stimulation of IL-6, TNF-, and IL-1ra release and subsequent activation of the HPA axis in postmenopausal women. These data show, for the first time in the human, that a physiological dose of estrogen can restrain cytokine and neuroendocrine responses to an inflammatory challenge in vivo. Inflammatory cytokines have been implicated in the pathogenesis of autoimmune and inflammatory diseases, as well as osteoporosis and cardiovascular disease (37, 38, 39). Within the brain, cytokine interactions have also been implicated in the pathogenesis of head injury, AIDS dementia complex, and Alzheimer’s disease (40). Because a protective effect of estrogen has been demonstrated in a number of these conditions, it remains to be determined whether this may, in part, be related to changes in cytokine activity. Thus, the results of our study have relevance to the pathogenesis of a number of human diseases in which estrogen-induced changes in inflammatory cytokine activity have been implicated.

Acknowledgments

We thank Dr. Michel Ferin for performing the E2 RIAs and Mrs. Irene Conwell for excellent technical assistance.

Footnotes

¹ This work was supported by NIH Grant MH-55708 and RR-00645 (to the Columbia General Clinical Research Center).

Received November 14, 2000.

Revised February 12, 2001.

Accepted March 1, 2001.

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