Estrogen receptor-alpha mediates the brain antiinflammatory activity of estradiol

Beyond the key role in reproductive and cognitive functions, estrogens have been shown to protect against neurodegeneration associated with acute and chronic injuries of the adult brain. Current hypotheses reconcile this activity with a direct effect of 17beta-estradiol (E2) on neurons. Here we demonstrate that brain macrophages are also involved in E2 action on the brain. Systemic administration of hormone prevents, in a time- and dose-dependent manner, the activation of microglia and the recruitment of peripheral monocytes induced by intraventricular injection of lipopolysaccharide. This effect occurs by limiting the expression of neuroinflammatory mediators, such as the matrix metalloproteinase 9 and lysosomal enzymes and complement C3 receptor, as well as by preventing morphological changes occurring in microglia during the inflammatory response. By injecting lipopolysaccharide in estrogen receptor (ER)-null mouse brains, we demonstrate that hormone action is mediated by activation of ERalpha but not of ERbeta. The specific role of ERalpha is further confirmed by comparing the effects of ERs on the matrix metalloproteinase 9 promoter activity in transient transfection assays. Finally, we report that genetic ablation of ERalpha is associated with a spontaneous reactive phenotype of microglia in specific brain regions of adult ERalpha-null mice. Altogether, these results reveal a previously undescribed function for E2 in brain and provide a mechanism for its beneficial activity on neuroinflammatory pathologies. They also underline the key role of ERalpha in brain macrophage reactivity and hint toward the usefulness of ERalpha-specific drugs in hormone replacement therapy of inflammatory diseases.     

Estrogen receptor-alpha mediates the protective effects of estrogen against vascular injury

Blood vessel cells express the 2 known estrogen receptors, alpha and beta (ERalpha, ERbeta), which are thought to mediate estrogen inhibition of vascular injury and atherosclerosis, but the relative role of ERalpha and ERbeta in these events is controversial. Estrogen inhibits the vascular injury response to the same extent in ovariectomized female wild-type mice and in the original single gene knockout mice for ERalpha (ERalphaKO(Chapel Hill) [ERalphaKO(CH)]) and ERbeta (ERbetaKO(Chapel Hill) [ERbetaKO(CH)]). In double gene knockout mice generated by crossing these animals (ERalpha,betaKO(CH)), estrogen no longer inhibits medial thickening after vascular injury, but still inhibits vascular smooth muscle cell proliferation and increases uterine weight. The partial retention of estrogen responsiveness in ERalpha,betaKO(CH) mice could be due either to the presence of a novel, unidentified estrogen receptor or to functional expression of an estrogen receptor-alpha splice variant in the parental ERalphaKO(CH) mice. To distinguish between these possibilities, we studied recently generated mice fully null for estrogen receptor alpha (ERalphaKO(Strasbourg) [ERalphaKO(St)]) and examined the effect of estrogen on the response to vascular injury. In the present study, we show that after vascular injury in ovariectomized ERalphaKO(St) mice, estrogen has no detectable effect on any measure of vascular injury, including medial area, proteoglycan deposition, or smooth muscle cell proliferation. These data demonstrate that estrogen receptor-alpha mediates the protective effects of estrogen on the response to vascular injury.     

Interleukin 1 beta mediates stress-induced immunosuppression via corticotropin-releasing factor.

Intracerebroventricular (icv) infusion of human interleukin 1 beta (IL-1) into intact and adrenalectomized rats impairs immune function. Using antibody to IL-1 as well as an inhibitor of IL-1 action, we sought to determine if endogenous IL-1 in the central nervous system has a physiological role in mediating the immunosuppressive effects of stress. Compared with freely moving controls, rats given intermittent electric shock to the tail for 40 min exhibited a fall in T lymphocyte proliferation and natural killer (NK) cell cytotoxicity of 33% and 38%, respectively; however, when pretreated with icv human IL-1 monoclonal antibody, which significantly crossreacts with rat IL-1, the decrement was attenuated to 14.6% and 15%, respectively. When rats were pretreated with icv alpha-MSH, which blocks many IL-1 effects, shock-induced suppression of 42% in both T lymphocyte proliferation and NK cytotoxicity were blunted to 33% and 31%, respectively. Similar results were found in adrenalectomized rats. These findings suggest that endogenous IL-1 is a physiologically relevant mediator of the immune response to stress. As IL-1 has been reported to release CRF, which we have shown always plays a significant role in stress-induced immunomodulation, we then assessed the relationship of IL-1 and CRF in immunosuppression. Infusion of icv IL-1 caused a decrease of 35% in T lymphocyte proliferation and 34% in NK activity, but pretreatment with CRF antibody icv attenuated IL-1 suppression of T lymphocyte proliferation and NK activity to 10% and 8%, respectively. Comparable results were observed in adrenalectomized rats. These findings suggest that CRF antibody is able to block the immunosuppressive effects of IL-1. To further examine the interaction of CRF in mediating stress-induced immunosuppression, we found that animals pretreated with icv CRF antibody, shocked and then given icv IL-1, had a decrement in T lymphocyte proliferation and NK cytotoxicity of 24% and 21%, respectively, demonstrating that the immunosuppressive effect of icv IL-1 is blocked when central CRF has been neutralized by prior administration of icv CRF antibody. In contrast, animals pretreated with icv IL-1 antibody, shocked and then given icv CRF, had decrements of 38% and 40%, respectively, showing that icv CRF does act even when central IL-1 has been neutralized by prior administration of icv IL-1 antibody. Thus, we conclude there is a sequential relationship between two of the known mediators of stress-induced immunosuppression, with release of central IL-1 followed by that of CRF.     

Estrogen receptor-alpha mediates the epidermal growth factor-stimulated prolactin expression and release in lactotrophs

Epidermal growth factor (EGF) is a potent regulator of cell function in many cell types. EGF-receptor (EGFR/ErbB1)-activated Erk1/2 has been reported to activate estrogen receptor (ER) in an estrogen (E2)-independent manner. In the pituitary lactotrophs, both EGF and E2 stimulate prolactin (PRL) release, but the nature of interactions between ErbB and ERalpha signaling is unknown. Our objectives were to 1) characterize EGF-induced PRL release, 2) determine whether this effect requires ERalpha, and 3) determine the molecular basis for cross talk between ErbB and ERalpha signaling pathways. Using GH3 cells, a rat lactotroph cell line, we report that EGF stimulates PRL gene expression and release in a dose- and time-dependent manner. EGF caused a rapid and robust activation of Erk1/2 via ErbB1 and induced phosphorylation of S118 on ERalpha in an Erk1/2-dependent manner. The global antiestrogen ICI 182780 and the ERalpha-specific antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylet hoxy)phenol]-1H-pyrazole dihydrochloride (MPP), but not the ERbeta-specific antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), blocked the EGF-induced PRL release, indicating an ERalpha requirement. This was further supported by using ERalpha knockdown by small interfering RNA. Because the antiestrogens did not block EGF-induced Mek-1 or Erk1/2 phosphorylation, ERalpha is placed downstream from the ErbB1-activated Erk1/2. These results provide the first evidence that ErbB1-induced PRL release is ERalpha dependent.     

Estrogen receptor-beta mediates dihydrotestosterone-induced stimulation of the arginine vasopressin promoter in neuronal cells

Arginine vasopressin (AVP) is a neuropeptide involved in the regulation of fluid balance, stress, circadian rhythms, and social behaviors. In the brain, AVP is tightly regulated by gonadal steroid hormones in discrete regions with gonadectomy abolishing and testosterone replacement restoring normal AVP expression in adult males. Previous studies demonstrated that 17beta-estradiol, a primary metabolite of testosterone, is responsible for restoring most of the AVP expression in the brain after castration. However, 5alpha-dihydrotestosterone (DHT) has also been shown to play a role in the regulation of AVP expression, thus implicating the involvement of both androgen and estrogen receptors (ER). Furthermore, DHT, through its conversion to 5alpha-androstane-3beta,17beta-diol, has been shown to modulate estrogen response element-mediated promoter activity through an ER pathway. The present study addressed two central hypotheses: 1) that androgens directly modulate AVP promoter activity and 2) the effect is mediated by an estrogen or androgen receptor pathway. To that end, we overexpressed androgen receptor, ERbeta, and ERbeta splice variants in a neuronal cell line and measured AVP promoter activity using a firefly luciferase reporter assay. Our results demonstrate that DHT and its metabolite 5alpha-androstane-3beta,17beta-diol stimulate AVP promoter activity through ERbeta in a neuronal cell line.     

Estrogen receptor beta mediates gender differences in ischemia/reperfusion injury

Under hypercontractile conditions associated with increased intracellular calcium, male hearts show enhanced ischemia/reperfusion injury compared to female hearts. Our aim in this study was to identify the specific estrogen receptor involved in this gender difference. Following brief treatment with isoproterenol, isolated mouse hearts were subjected to ischemia and reperfusion. Postischemic contractile function and infarct size were measured in wild-type (WT) male and female hearts, and female hearts lacking functional alpha estrogen receptor (alpha ERKO), or the beta estrogen receptor (beta ERKO). WT male hearts exhibited significantly less functional recovery and more necrosis than WT females. alpha ERKO female hearts exhibited ischemia/reperfusion injury similar to that observed in WT females, whereas beta ERKO females exhibited significantly less functional recovery than WT females and were similar to WT males. These data suggest that estrogen, through the beta-estrogen receptor, plays a role in the protection observed in the female heart. Furthermore, we identified genes that were differentially expressed in beta ERKO female hearts compared to alpha ERKO and WT female hearts, and found altered expression of a number of metabolism genes, which may be important in ischemic injury. We further showed that WT female hearts have increased ratio of carbohydrate to fatty acid metabolism relative to WT males.     

IL-10 mediates immunosuppression following primary infection with Toxoplasma gondii in mice

Suppression of the host immune response by Toxoplasma gondii has been observed in both human and experimental murine infection. In this study, inbred mice were infected with T. gondii. At day 7 post-infection, the lymphoproli-ferative response to both mitogen and superantigen as well as parasite antigen were found to be significantly depressed. Using a transwell system, it was determined that the reduced proliferative response was due to soluble factor (s) being expressed by splenocytes from the infected mice. Isolation of the splenocytes into an adherent and nonadherent population suggested that both macrophages and T cells were able to produce at least one soluble factor. Tissue culture supernatant derived from the splenocytes of the infected mice contain increased levels of IL-10, whereas measurable IL-2 levels could not be quantitated. At day 7 post-infection, both a biologic assay for IFN-γ in culture supernatant and the expression of IFN-γ mRNA in the splenocytes were reduced. Antibody to IL-10 was able to partially neutralize (almost 50%) the in vitro immune downregulation of the tissue culture supernatant. Anti-IL-10 in combination with a nitric oxide (NO) antagonist was able to reverse the inhibitory activity of the culture supernatant by 85%. Since IL-10 is a potent antagonist of IFN-γ, it may represent a critical cytokine involved in mediating T. gondii induced immunosuppression in the infected host.     

Age-related changes in sex hormones affect the sex difference in serum leptin independently of changes in body fat

Serum leptin concentrations are highly correlated with body fatness, but there is considerable variability among individuals after adjusting for differences in body fatness. Theoretically, sex hormone levels may influence serum leptin, since the levels are higher in women than in men independently of body fat. Increasing old age is associated with decreases in serum sex hormone concentrations and changes in body fatness that may independently alter serum leptin concentrations. In a cross-sectional sample of 106 men and 166 women aged 62 to 98 years, serum leptin adjusted for total body fat had a significant positive association with age in men and a nonsignificant negative association with age in women. Serum testosterone had a significant negative association with serum leptin in men after adjusting for total body fat, the fasting insulin resistance index (FIRI), and sex hormone-binding globulin (SHBG). In a longitudinal sample of 22 elderly men and 52 women, serum leptin levels increased significantly over a 14-year period in men, but not in women. Increases in serum leptin were significantly associated with decreases in serum testosterone but not with changes in the body mass index (BMI) in men. In contrast, changes in leptin were associated with changes in the BMI but not with changes in serum estrone in women. These results suggest that differences among men and changes with age in serum leptin are associated with circulating levels of testosterone. Elderly men become progressively "hyperleptinemic" with age regardless of changes in body fatness, possibly due to decreasing testosterone levels.     

A major sex difference in kallikrein-like activity in the rat anterior pituitary

Male and female rat pituitaries were compared for their ability to release kinins from kininogen at pH 8.5 (kininogenase activity) and cleave chromogenic peptide substrates for various proteases at pH 8.0. The female anterior pituitary was found to contain over 18 times more kininogenase activity than the female anterior pituitary. The neurointermediate lobe did not exhibit a significant sex difference in kininogenase activity. The kininogenase of the female anterior pituitary was sensitive to inhibition by aprotinin, but was resistant to soybean trypsin inhibitor. Female anterior pituitary homogenates also cleaved chromogenic peptide substrates for kallikrein over 15 times faster than male anterior pituitary homogenates. The results indicate a major sex difference in kallikrein-like activity in the rat anterior pituitary.     

A sex difference in the hemodynamic effects of chronic normobaric hypoxia in rats

The packed cell volume was higher and the increase in systolic pressure in the right ventricle, as an indication of systolic pressure in the pulmonary artery, was smaller in female rats previously exposed for 4 weeks to normobaric hypercapnic hypoxia, as compared to males treated in the same way. Gonadectomy performed in male rats either on the first day of life or after maturation did not have a significant influence on circulatory reaction to chronic normobaric hypoxia. Female rats in permanent estrus and those with ovariectomy had larger posthypoxic hypertrophy of the right ventricle as indicated by higher values for right/left ventricular weight ratio and relative weight of the right ventricle, than untreated controls.