Cortisol response and immune-related effects of Atlantic salmon (Salmo salar Linnaeus) subjected to short- and long-term stress

It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon (Salmo salar Linnaeus) the effects of both short- (a single 15s out of water) and long-term (4 weeks of daily handling 15s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1beta (IL-1beta) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6h post stress. Constitutive expression of IL-1beta in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1beta in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1 week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1beta expression in macrophages isolated from stressed fish over the first 2 weeks. At weeks 1, 2 and 3 the magnitude of IL-1beta response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4 weeks there was no significant difference in inducible IL-1beta expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1beta, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased leukocyte survival when exposed to A. salmonicida. The implications of these changes in the immune system will be discussed with respect to the use of classical indicators of stress to predict possible effects on the immune system of fish.     

IL-1beta stimulates alveolar fluid absorption in fetal guinea pig lungs via the hypothalamus-pituitary-adrenal gland axis

We tested the hypothesis that interleukin (IL)-1beta-induced cortisol synthesis stimulates alveolar fluid clearance in preterm fetuses. IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. Fetuses were obtained by abdominal hysterotomy at 61 and 68 days gestation and instilled with isosmolar 5% albumin in the lungs, and alveolar fluid movement was measured over 1 h from the change in alveolar protein concentration. Alveolar fluid clearance was induced at 61 days gestation and stimulated at 68 days gestation by IL-1beta, which both were attenuated by cortisol synthesis inhibition. Plasma ACTH and cortisol concentrations were increased by IL-1beta at both gestational ages, and metyrapone reduced cortisol concentrations. IL-1beta was mostly low or undetectable in both fetal and maternal blood. Prenatal alveolar fluid clearance, when present as well as IL-1beta induced, was always propranolol and amiloride sensitive, suggesting that beta-adrenoceptor stimulation and amiloride-sensitive Na+ channels were critical for fluid absorption. IL-1beta increased lung beta-adrenoceptor density at gestation day 61, and cortisol synthesis inhibition attenuated the increased beta-adrenoceptor density. Epithelial Na+ channel and Na+-K+-ATPase subunit expressions were both increased by IL-1beta and attenuated by cortisol synthesis inhibition. These results may explain why babies delivered preterm after intrauterine inflammation have a reduced risk of developing severe respiratory distress.     

Allergen rush immunotherapy increases interleukin (IL)-12 production and IL-12 receptor beta2 chain expression in patients with allergic asthma

Interleukin (IL)-12 production and IL-12 receptor (IL-12R) beta2 chain expression were investigated in patients with allergic asthma successfully treated with rush immunotherapy (RIT) and control patients with mild allergic asthma. Peripheral blood mononuclear cells (PBMCs) were stimulated with Dermatophagoides farinae (Der f), and production of cytokines was measured. Furthermore, the effects of cytokines on IL-12R beta2 chain expression on CD4(+) T cells were investigated. Production by PBMCs of IL-12 and IFN-gamma was significantly higher and production of IL-4 was significantly lower after stimulation with Der f allergen in RIT-treated patients than in control patients. Significant increases in the expression of IL-12R beta2 chain before and after stimulation of CD4(+) T cells with IL-12 or IFN-gamma were observed in RIT-treated patients compared with that in control patients. Allergen RIT increases IL-12 production and IL-12R beta2 chain expression and thus may convert cytokine production from Th2 to Th1 or Th0 type in allergic asthma.     

Not in the mind of neurasthenic lazybones but in the cell nucleus: patients with chronic fatigue syndrome have increased production of nuclear factor kappa beta

There is now some evidence that chronic fatigue syndrome is accompanied by an activation of the inflammatory response system and by increased oxidative and nitrosative stress. Nuclear factor kappa beta (NFkappabeta) is the major upstream, intracellular mechanism which regulates inflammatory and oxidative stress mediators. In order to examine the role of NFkappabeta in the pathophysiology of CFS, this study examines the production of NFkappabeta p50 in unstimulated, 10 ng/mL TNF-alpha (tumor necrosis factor alpha) and 50 ng/mL PMA (phorbolmyristate acetate) stimulated peripheral blood lymphocytes of 18 unmedicated patients with CFS and 18 age-sex matched controls. The unstimulated (F=19.4, df=1/34, p=0.0002), TNF-alpha-(F=14.0, df=1/34, p=0.0009) and PMA-(F=7.9, df=1/34, p=0.008) stimulated production of NFkappabeta were significantly higher in CFS patients than in controls. There were significant and positive correlations between the production of NFkappabeta and the severity of illness as measured with the FibroFatigue scale and with symptoms, such as aches and pain, muscular tension, fatigue, irritability, sadness, and the subjective feeling of infection. The results show that an intracellular inflammatory response in the white blood cells plays an important role in the pathophysiology of CFS and that previous findings on increased oxidative stress and inflammation in CFS may be attributed to an increased production of NFkappabeta. The results suggest that the symptoms of CFS, such as fatigue, muscular tension, depressive symptoms and the feeling of infection reflect a genuine inflammatory response in those patients. It is suggested that CFS patients should be treated with antioxidants, which inhibit the production of NFkappabeta, such as curcumin, N-Acetyl-Cysteine, quercitin, silimarin, lipoic acid and omega-3 fatty acids.     

硬膜外复合全身麻醉对开胸手术患者血流动力学和应激反应的影响研究

目的:研究硬膜外复合全身麻醉对开胸手术患者血流动力学和应激反应的影响。方法:选取120例我院2015年1月到2016年1月收治的开胸手术患者,按照随机数字表法将患者分为研究组和对照组,对照组给予单纯全麻,研究组给予硬膜外复合全身麻醉,比较两组苏醒时间、苏醒期收缩压、舒张压、心率及烦躁情况,并比较两组术前、术后72 h血糖、皮质醇以及肾上腺素水平。结果:两组苏醒时间比较无统计学意义(P0.05),苏醒期研究组收缩压、舒张压及心率、烦躁发生率均较对照组降低(P0.05);两组术前血糖、皮质醇及肾上腺素比较无统计学差异(P0.05);术后72 h,两组血糖、皮质醇及肾上腺素均升高(P0.05);研究组血糖、皮质醇及肾上腺素均较对照组降低(P0.05)。结论:硬膜外复合全身麻醉应用于开胸手术能显著提高患者苏醒期苏醒质量,稳定血流动力学,改善其应激反应。     

Influence of surgery on in-vitro cytokine production by human monocytes

Surgery leads to significant modulation of the immune system, in which cytokines play a major role. Circulating interleukin 6 (IL-6) and IL-1 have been reported following surgery whereas tumor necrosis factor alpha (TNF-alpha) is only found in gut ischemia-associated surgery. We have investigated the consequences of surgery on in-vitro cytokine production by human monocytes stimulated by lipopolysaccharide (LPS) and staphylococcal toxic shock syndrome toxin-1 (TSST-1). Comparisons were made between the responsiveness of cells obtained the day before (D-1), during (D0) and after (D1, D2, D3) surgery. Patients undergoing abdominal aortic surgery (N = 9), carotid surgery (N = 4) and spinal surgery (N = 4) have been studied. A significant decrease of TNF-alpha, IL-1 beta and IL-1 alpha production by monocytes prepared from blood samples taken during the surgery was noticed, whereas IL-6 production was not significantly modified. On D2 a significant increase of monocyte responsiveness was observed and levels of cytokine productions rose back to initial values by the end of the follow up. The diminished in-vitro cytokine production observed during surgery might be the consequence of the effects of anaesthetic drugs, whereas the enhancement observed on D2 might reflect the surgical stress, leading to in-vivo priming of circulating monocytes.     

Dynamics of cytokine production in human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia

To specify the role of individual cytokines in the immune response to pyrogens, isolated and cultivated human peripheral blood mononuclear cells (PBMC) were used for the experiments. Different pyrogens (lipopolysaccharide from Escherichia coli - LPS and live Borrelia afzelii) were applied and the time course of changes in concentrations of different cytokines in the medium was followed using the ELISA method. It was found that nonstimulated human PBMC proliferate under in vitro conditions and produce IL-6, TNF-alpha, IL-10 and finally also IL-1beta. Productions of IL-12 and INF-gamma are not changed. Proliferation of PBMC is potentiated after incubation with LPS or live Borrelia. PBMC stimulated by LPS increase the net production (stimulated minus unstimulated) of IL-1beta and TNF-alpha significantly, while production of IL-6 was smaller. A delayed increase in the production of IL-10 was also observed. Productions of IL-12 and INF-gamma were not influenced. In contrast to LPS, stimulation of PBMC with live Borrelia, increases also the production of IL-12 and IFN-gamma, besides IL-1beta, TNF-alpha, IL-6 and IL-10. Productions of IL-1beta, IL-6 and TNFalpha increased immediately after incubation with both LPS and Borrelia, while productions of IL-12 and INF-gamma begin to increase 8 hours and production of IL-10 12 hours after stimulation. Data indicate that stimulation with different pyrogens may activate the cells of the immune cascade in a different way. Stimulation of BPMC by LPS seems to activate the initial steps of the immune response (macrophages and granulocytes) only, while infection with live Borrelia also stimulates the later phase of the immune response, probably due to effect of initially produced cytokines.     

Dynamics of cytokine production in human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia

To specify the role of individual cytokines in the immune response to pyrogens, isolated and cultivated human peripheral blood mononuclear cells (PBMC) were used for the experiments. Different pyrogens (lipopolysaccharide from Escherichia coli - LPS and live Borrelia afzelii) were applied and the time course of changes in concentrations of different cytokines in the medium was followed using the ELISA method. It was found that nonstimulated human PBMC proliferate under in vitro conditions and produce IL-6, TNF-alpha, IL-10 and finally also IL-1 beta. Productions of IL-12 and INF-gamma are not changed. Proliferation of PBMC is potentiated after incubation with LPS or live Borrelia. PBMC stimulated by LPS increase the net production (stimulated minus unstimulated) of IL-1 beta and TNF-alpha significantly, while production of IL-6 was smaller. A delayed increase in the production of IL-10 was also observed. Productions of IL-12 and INF-gamma were not influenced. In contrast to LPS, stimulation of PBMC with live Borrelia, increases also the production of IL-12 and IFN-gamma, besides IL-1 beta, TNF-alpha, IL-6 and IL-10. Productions of IL-1 beta, IL-6 and TNF alpha increased immediately after incubation with both LPS and Borrelia, while productions of IL-12 and INF-gamma begin to increase 8 hours and production of IL-10 12 hours after stimulation. Data indicate that stimulation with different pyrogens may activate the cells of the immune cascade in a different way. Stimulation of BPMC by LPS seems to activate the initial steps of the immune response (macrophages and granulocytes) only, while infection with live Borrelia also stimulates the later phase of the immune response, probably due to effect of initially produced cytokines.     

Depression of plasma levels of cytokines and ex-vivo cytokine production in relation to the activity of the pituitary-adrenal axis, in patients undergoing major vascular surgery.

The relation between the immune and neuroendocrine response during surgery was studied. In 18 patients undergoing major vascular surgery, circulating interleukin (IL)-1beta and ex-vivo production of IL-1beta and tumour necrosis factor (TNF)-alpha were lower on day 1 after surgery compared to pre-operation values (-14+/-5%, P<0.05; -62+/-9%, P<0.05; and -31+/-54%, P<0.005, respectively). Circulating IL-1 receptor antagonist (IL-1ra) was higher on the 5th day post-operatively compared to pre-operation values (mean +640%+/-400, P<0.05).In a more detailed study in six patients, the ex-vivo production of IL-1beta and TNF-alpha started to decrease at induction of general anaesthesia and dropped to under 10% of initial values at the end of surgery. Circulating IL-1ra and ex-vivo production of IL-1ra started to increase at the end of surgery and remained elevated up to 6 days post-operatively. Plasma antidiuretic hormone (ADH) and adrenocorticotropic hormone (ACTH) increased during surgery, but cortisol remained unchanged.We demonstrate a depression of circulating pro-inflammatory IL-1beta and an increase of circulating anti-inflammatory IL-1ra during surgical stress. The ex-vivo production of IL-1beta and TNF-alpha was suppressed, indicating a downregulation of the production of these cytokines. This parallelled the hormonal reaction with high ADH and ACTH, but not of cortisol, suggesting that glucocorticoid is not the key-factor in downregulation of production and release of pro-inflammatory cytokines.     

High self-perceived stress and many stressors, but normal diurnal cortisol rhythm, in adults with ADHD (attention-deficit/hyperactivity disorder)

Attention-deficit/hyperactivity disorder (ADHD) in adults is associated with significant impairment in many life activities and may thus increase the risk of chronic stress in everyday life. We compared adults with a DSM-IV ADHD diagnosis (n = 28) with healthy controls (n = 28) regarding subjective stress and amounts of stressors in everyday life, diurnal salivary cortisol in the everyday environment and salivary cortisol before and after cognitive stress in a laboratory setting. The association between cortisol concentrations and impulsivity was also investigated. Consistent with assumptions, individuals with ADHD reported significantly more self-perceived stress than controls, and subjective stress correlated with the amount of stressors in everyday life. The two groups were comparable with respect to overall diurnal cortisol levels and rhythm, as well as in pre- and post-stress cortisol concentrations. Post-stress cortisol (but not baseline cortisol) concentration was positively correlated with impulsivity. The group with high post-stress cortisol also reported more symptoms of depression and anxiety, as well as self-perceived stress and stressors in every-day life. The diagnosis of ADHD significantly increased the risk of belonging to the group with high post-stress cortisol levels. The results in this study warrant a focus not only on the primary diagnosis of ADHD, but also calls for a broader assessment of stressors and subjective stress in everyday life, as well as support comprising stress management and coping skills.     

Comparison of cytokine production in cultures of whole human blood and purified mononuclear cells.

In order to examine inter- and intra-individual variations in cytokine production, blood was collected from 48 healthy subjects on each of 4 occasions separated by 4 weeks. Whole blood (diluted 1:10) and mononuclear cell (MNC) cultures were stimulated for 24 h with either concanavalin A (Con A) or bacterial lipopolysaccharide (LPS) and the concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha, IL-10 and IFN-gamma in the culture medium measured. There were highly significant inter-individual variations in the production of each of the cytokines measured. However, the level of the production of each cytokine appeared to be characteristic of an individual. There were significant correlations between production of each cytokine in whole blood and MNC cultures. It is concluded that there is significant inter-individual variation in cytokine production which is unaffected by time or by the stimulus used to elicit cytokine production, and that whole blood cultures can be used instead of MNC cultures to measure cytokine production.     

Effect of a four-week course of interleukin-10 on cytokine production in a placebo-controlled study of HIV-1-infected subjects

Interleukin (IL)-10 suppresses synthesis of the pro-inflammatory cytokines tumor necrosis factor (TNF)alpha, IL-1beta, and interferon (IFN)gamma. Since pro-inflammatory cytokines have been implicated in the production of human immunodeficiency virus type 1 (HIV-1), cytokine synthesis in whole blood cultures were determined during a 4-week course of subcutaneous IL-10 injections in 33 HIV-1-infected patients. Patients were randomized into four groups: placebo (nine), IL-10 at 1 microg/kg/day (nine), IL-10 at 4 microg/kg/day (six) and IL-10 at 8 microg/kg three times per week (nine). Whole blood was obtained at the beginning and conclusion of the study and was stimulated for 24 hours with the combination of IL-18 plus lipopolysaccharide. TNFalpha production in stimulated whole blood was reduced three and six hours after the first injection of IL-10 compared to subjects injected with the placebo. After four weeks of treatment, production of IFNgamma was suppressed in a greater number of patients in the IL-10 treatment groups compared to subjects in the placebo group. Similarly, IL-1beta production was lower in the IL-10 treatment groups compared to subjects receiving placebo. In contrast, after four weeks of IL-10, circulating levels of the anti-inflammatory TNF soluble receptor p55 increased dose-dependently compared to placebo subjects. Patient heterogeneity and small sample size presented difficulties in establishing statistical significance. Although the cytokine changes in our study did not demonstrate statistically significant changes, the data nevertheless reveal that four weeks of IL-10 therapy in HIV-1 infected subjects produced the anticipated suppression of pro-inflammatory cytokines.     

Sleep impairments in healthy seniors: roles of stress, cortisol, and interleukin-1 beta

Study Objectives: Increased stress responsivity and a longer-lasting glucocorticoid increase are common findings in aging studies. Increased cortisol levels at the circadian nadir also accompany aging. We used 24h free urine cortisol to assess these age changes in healthy seniors. We hypothesized that free cortisol levels would explain individual differences in age-related sleep impairments. Design: The study compared sleep, cortisol, and sleep-cortisol correlations under baseline and “stress” conditions in men and women. Setting: Subjects were studied in the General Clinical Research Center under baseline conditions and a mildly stressful procedure (24h indwelling intravenous catheter placement). Participants: Eighty-eight healthy, nonobese subjects (60 women and 28 men) from a large study of successful aging participated in the study. Mean ages were 70.6 (±6.2) and 72.3 (±5.7) years for women and men, respectively. Measurements: The 24h urines were collected for cortisol assay (radioimmunoassay [RIA]); blood was sampled at three diurnal time points for assay (enzyme-linked immunosorbent assay [ELISA]) of interleukin-1 (IL-1) beta; sleep architecture and sleep electroencephalograms (EEGs) were analyzed (after an adaptation and screening night) on baseline and stress nights via polysomnography and EEG power spectral analysis. Results: Healthy older women and men with higher levels of free cortisol (24h urine level) under a mild stress condition had impaired sleep (lower sleep efficiency; fewer minutes of stages 2, 3, and 4 sleep; more EEG beta activity during non-rapid eye movement sleep [NREM] sleep). Similar results were obtained when stress reactivity measures were used (cortisol and sleep values adjusted for baseline values), but not when baseline values alone were used. Gender differences were apparent: Men had higher levels of free urine cortisol in both baseline and mild stress conditions. Cortisol and sleep correlated most strongly in men; cortisol stress response levels explained 36% of the variance in NREM sleep stress responses. In women, but not men, higher cortisol was also associated with earlier time of arising and less REM sleep. Higher cortisol response to stress was associated with increased circulating levels of IL-1β, explaining 24% of the variance in a subset of women. Conclusion: These results indicate that free cortisol (as indexed by 24h urine values) can index responses to mild stress in healthy senior adults, revealing functional correlations (impaired sleep, earlier times of arising, more EEG beta activity during sleep, more IL-1β) and gender differences. (Chronobiology International, 17(3), 391-404, 2000)     

Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-alpha or IL-1beta. IFN-gamma as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-alpha induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-alpha also inhibited the TNF-alpha-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1beta or TNF-alpha alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.     

Changes in plasma IL-1beta, TNF-alpha and IL-6 after total hip replacement surgery in general or regional anaesthesia

Different anaesthetic methods influence the neuro-immuno-endocrine biologic responses to surgery and may thus possibly interfere with the postoperative course and development of complications. The neuroendocrine system is closely related to the cytokine network. In this study, the effects of general anaesthesia (n=6) and regional spinal/epidural anaesthesia (n=6) on the cytokine response (IL-1beta, TNFalpha, IL-6) to uncemented total hip replacement surgery were evaluated. The postoperative clinical course was uneventful in every case. In both groups, only very low values of plasma IL-beta were measured perioperatively, whereas plasma IL-6 increased postoperatively with peak values 4 h after surgery. The changes in plasma TNF-alpha were not significant. No significant differences in plasma TNF-alpha or IL-6 were found between patients operated in general or in regional anaesthesia. This suggests minor influence of plasma cytokines on the possible beneficial effects of regional anaesthesia on the clinical course after surgery in low risk patients. There were slightly higher TNF-alpha and IL-6 levels after the operation and significantly lower cortisol levels during the operation in the regional anaesthesia group compared to the general anaesthesia group, giving rise to a significant inverse correlation between peak values of IL-6 and peak values of cortisol. This supports the theory that after surgery the inhibitory effect of cortisol on monocyte cytokine production overrides adrenergic stimulation.     

Oxytocin-induced labor augments IL-1beta-stimulated lung fluid absorption in fetal guinea pig lungs

We tested the hypothesis that oxytocin-induced labor augmented IL-1beta-induced/-stimulated lung fluid absorption in preterm guinea pig fetuses. IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. At day 3, oxytocin was administered, and fetuses were delivered by abdominal hysterotomy at 61 and by oxytocin-induced birth at 68 days gestation. Delivered fetuses were instilled with isosmolar 5% albumin into the lungs, and lung fluid movement was measured over 1 h by mass balance. Lung fluid absorption was induced in 61-day and stimulated in 68-day gestation lungs by IL-1beta. Labor induction by oxytocin augmented IL-1beta-induced/-stimulated lung fluid absorption. Metyrapone pretreatment did not affect oxytocin-induced/-stimulated lung fluid absorption, while completely blocking IL-1beta-induced/-stimulated fluid absorption. Fetal lung fluid absorption, when present, was always propranolol and amiloride sensitive, suggesting that beta-adrenoceptor stimulation and amiloride-sensitive sodium channels were critical for fluid absorption. Epithelial sodium channel and Na-K-ATPase subunit expressions were both increased by IL-1beta, but not further by oxytocin. Our results indicate that IL-1beta release into the maternal blood circulation positively affects lung maturation due to the IL-1beta-induced release of cortisol and thus prepares the lungs for the epinephrine surge associated with labor.     

Dynamic measure of production rate of cortisol in the mature guinea-pig in response to the stress of anesthesia: Effect of estradiol

The effect of estradiol on adrenal secretion rate of cortisol in response to a stress induced by anesthesia, was examined by comparing the metabolic clearance rate and the production rate of cortisol between males and females and after estradiol administration in castrated animals. Metabolic clearance rates of cortisol (MCR) were significantly higher (+30%) in males than in females. Castration lowered the MCR of cortisol in males and had no significant effect in females. After estradiol administration, a fall in the MCR of cortisol concomitant with a rise in blood cortisol level was observed especially in males in which the effect of treatment was more marked than in females and highly significant. The production rate of cortisol was identical in males and females and was slightly increased in estradiol-treated males and females. The data indicate that estradiol had an inhibitory effect on metabolic clearance of cortisol, which caused an important rise of blood cortisol levels in response to stress and which prevented an increase in the adrenal response to the stress. Since the pituitary adrenal cortex can respond in a normal way to stress, the low value of MCR of cortisol could be the limiting factor in the adrenal secretion rate of cortisol in estrogen-treated guinea-pigs.