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Association of Interleukin-8 with Hot Flashes in Premenopausal, Perimenopausal, and Postmenopausal Women and Bilateral Oophorectomized Women

Departments of Obstetrics and Gynecology (T.U., H.U., J.T., Y.M., M.Ya., A.K., T.M., M.M., M.I.) and Preventive Medicine (H.U.), Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan; and Pharmacology Research Department (N.T., M.Yu., S.T.), Tsumura Central Research Institute, Ibaraki 300-1192, Japan

Address all correspondence and requests for reprints to: Toshiyuki Yasui, Department of Obstetrics and Gynecology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan. E-mail: yasui{at}clin.med.tokushima-u.ac.jp.

	Abstract

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Objective: The purpose of this study was to identify serum cytokine concentrations in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women with hot flashes.

Methods: Serum concentrations of 17 cytokines were simultaneously measured using a multiplexed human cytokine assay in 129 premenopausal, perimenopausal, and postmenopausal women and 50 bilateral oophorectomized women.

Results: Serum IL-8 concentrations in midlife women and bilateral oophorectomized women with severe hot flashes were significantly higher than the concentrations in women without hot flashes and women with mild and moderate hot flashes. Serum macrophage inflammatory protein-1ß concentration in women with severe hot flashes was significantly higher than those in women without hot flashes and women with mild and moderate hot flashes.

Conclusion: Serum IL-8 concentrations in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women with hot flashes were significantly higher than those in women without hot flashes. IL-8 may be associated with peripheral vasodilation in women with hot flashes.

	Introduction

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HOT FLASHES AND night sweating are the most common medical complaints in women during the menopausal transition, and the prevalence of hot flashes or night sweating in Japanese and Chinese women has been found in a multiethnic study to be lower than that in Caucasian women (1, 2). It has also been reported that the prevalence of night sweating in Japanese women was significantly lower than that in Australian women (3). To date, the pathophysiology of hot flashes has been not elucidated. Peripheral vasodilation and increase in skin temperature observed in women with hot flashes might be induced by changes in brain neurotransmitters and instability in the hypothalamic thermoregulatory center due to estrogen withdrawal (4). In addition, an elevated level of calcitonin gene-related peptide, which is a potent vasodilator neuropeptide in sensory nerves, has been reported to be associated with increase in finger tip temperature (5).

Cytokines such as IL-6 and TNF- have been reported to be potent vasodilators (6, 7). Recently, it has also been reported that an increase in serum IL-8 concentration was associated with peripheral vasodilation in patients after coronary artery bypass grafting (8). It has been reported that serum levels of IL-6 and TNF- in postmenopausal women were higher than those in fertile women (9) and that the production of IL-6 was decreased in women receiving estrogen treatment (10). In addition, it has been reported that there was an inverse correlation between serum dehydroepiandrosterone or dehydroepiandrosterone-S levels and serum IL-6 concentrations (9). Because levels of cytokines are related to hormonal levels, cytokines may be associated with peripheral vasodilation in women with hot flashes induced by hormonal changes. However, a relationship between hot flashes and serum cytokine concentrations has not been reported.

The aim of this study was to identify the serum cytokine concentrations in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women with hot flashes.

	Subjects and Methods

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Subjects

The subjects of this study were recruited from women visiting the outpatient clinic of the Department of Obstetrics and Gynecology, Tokushima University Hospital. One hundred fifty midlife women and 50 bilateral oophorectomized women who visited the outpatient clinic for treatment of vasomotor symptoms or for screening of gynecological cancer were eligible for this study, and 129 midlife women and 50 bilateral oophorectomized women were enrolled in this study. Informed consent for participation in this study was obtained from each woman. The Ethics Committee of Tokushima University Hospital approved the study. Reviews of medical histories and the results of physical examinations and blood chemistry tests showed that all of the women were in good health. None of the subjects had taken any medication known to influence the immune system for at least 1 yr. Subjects suspected of having infectious diseases, inflammatory disorders, malignancy or autoimmune diseases, of being undernourished, or of abusing alcohol or drugs were excluded according to the SENIEUR protocol (11). According to the classification of menopausal status (12), the 129 women enrolled in this study included 16 premenopausal women who had a recent history of regular menstruation (25–35 d per cycle), 56 perimenopausal women who had experienced alterations in menstrual frequency and/or flow in the 12 months preceding entry into the study, and 57 postmenopausal women who had passed through a natural menopause at least 12 months before entry into the study. The 50 bilateral oophorectomized women had stopped menstruation because of surgical removal of the ovaries. Venous blood samples were drawn into tubes between 0800–1000 h. Blood samples were taken from premenopausal women during the follicular phase (d 5–9 of the menstrual cycle). Samples obtained were frozen at –70 C until use for analysis. The assay was performed at the same time.

A detailed history of hot flashes was determined by a series of questions. We assessed severity of hot flashes using the Food and Drug Administration published draft guidance for clinical evaluation of vasomotor symptoms (13). Severity is defined as mild (sensation of heat without sweating), moderate (sensation of heat with sweating, able to continue activity), and severe (sensation of heat with sweating, causing cessation of activity).

Measurement of cytokine concentrations

A Bio-Plex human cytokine 17-Plex assay kit (Bio-Rad Laboratories, Hercules, CA) was used to test samples for the presence of 17 cytokines: IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF-, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, IFN-, macrophage inflammatory protein (MIP)-1ß, and monocyte chemotactic protein-1. The assay was performed in a 96-well filtration plate supplied with the assay kit. Briefly, each serum sample was incubated with Bio-Plex cytokine microbeads for 30 min at room temperature. After washing three times, the beads were incubated with detection antibodies for 30 min. After three washes, the beads were combined with streptavidin-PE and incubated for 10 min. After washing, the beads were resuspended in assay buffer. Beads were read on the Bio-Plex suspension array system, and the data were analyzed using Bio-Plex Manager software version 3.0 with 5PL curve fitting. The intraassay and interassay coefficients of variation were 2.0–10.0% and 3.5–16.1%, respectively. The sensitivity levels were 1.1 pg/ml for IL-6 and MIP-1ß and 0.5 pg/ml for IL-8.

Measurements of estradiol

Serum estradiol concentration was measured by a two-site immunoenzymometric assay using a kit (TOSOH Co., Tokyo, Japan). The intraassay and interassay coefficients of variation were 4–9% and 6–9%, respectively, and the sensitivity of the assay was 5 pg/ml.

Statistical analysis

Results are presented as means ± SD values. One-way ANOVA and analysis of covariance were used to compare differences between groups. To determine differences between groups, Mann-Whitney or Kruskal-Wallis tests were used if data were not normally distributed. Correlations were determined by using Spearman’s rank order analysis and linear regression analysis. P values <0.05 were considered to be statistically significant.

	Results

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Background characteristics

As shown in Table 1, the 129 premenopausal, perimenopausal, and postmenopausal women and 50 bilateral oophorectomized women were divided into four groups (free, mild, moderate, and severe) according to the degree of hot flashes. Serum estradiol concentration in premenopausal, perimenopausal, and postmenopausal women with severe hot flashes was significantly lower than the concentrations in women without hot flashes and women with mild hot flashes.

As shown in Fig. 1, serum IL-8 concentration in premenopausal, perimenopausal, and postmenopausal women with severe hot flashes (101.5 ± 53.4 pg/ml) was significantly (P < 0.0001, P < 0.0001, and P < 0.01, respectively) higher than the concentrations in women without hot flashes and women with mild and moderate hot flashes (7.6 ± 6.3, 26.7 ± 33.2, and 54.4 ± 63.7 pg/ml, respectively). Serum IL-8 concentration in bilateral oophorectomized women with severe hot flashes (106.1 ± 50.2 pg/ml) was significantly (P < 0.0001, P = 0.0002, and P = 0.0004, respectively) higher than the concentrations in women without hot flashes and women with mild and moderate hot flashes (22.0 ± 21.3, 32.6 ± 45.2, and 33.3 ± 26.3 pg/ml, respectively). Serum MIP-1ß concentrations in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women with severe hot flashes were significantly higher than the concentrations in women without hot flashes and women with mild and moderate hot flashes. In addition, serum IL-6 concentrations in midlife women with moderate and severe hot flashes were significantly higher than the concentration in women without hot flashes. Serum IL-6 concentration in bilateral oophorectomized women with severe hot flashes was also significantly higher than the concentrations in women without hot flashes and women with mild hot flashes.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Associations of serum concentrations of IL-8, MIP-1ß, and IL-6 with degree of hot flashes in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women. Horizontal small bars represent the 10–90th percentile range, and boxes indicate the 25–75th percentile range. The horizontal line in each box corresponds to the median.

	Discussion

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It has been reported that cytokine-induced neutrophil chemoattractant, which is IL-8 in humans, was produced in the paraventricular hypothalamic nucleus and anterior pituitary gland in response to stress in the rat (14). Therefore, increase in IL-8 during hot flashes may be induced by the production and secretion from the hypothalamus and anterior pituitary gland through a decrease in estradiol level. The hypothalamic-pituitary-adrenal (HPA) axis is the key player in stress response (15). Hot flashes, as a stressor, induced by hormonal changes might stimulate the HPA axis. Recently, it has been reported that IL-8 mRNA was expressed in and that IL-8 was released from human adrenocortical cells (16). IL-8 may also be produced in and released from adrenocortical cells through stimulation of the HPA axis due to hot flashes. On the other hand, IL-8 is produced by macrophages, including macrophages in all layers of the human adrenal gland (17). It has been reported that the estrogen receptor was expressed in macrophages (18). IL-8 might be released from macrophages by a direct effect on macrophages of reduction in estradiol level. Serum estradiol concentrations gradually decreased in premenopausal, perimenopausal, and postmenopausal women, whereas serum estradiol concentrations abruptly decreased after bilateral oophorectomy. The difference in the changes in estradiol concentrations between midlife women and bilateral oophorectomized women may be associated with different effects on the production of IL-8. Further study is needed to determine the source of the secretion of IL-8.

The production of MIP-1ß has also been shown to be induced by various proinflammatory cytokines such as TNF- and IFN- (19). In the present study, the source of production of MIP-1ß in women with hot flashes is not clear. We demonstrated that serum MIP-1ß concentrations as well as serum IL-8 concentrations increased in midlife and bilateral oophorectomized women with severe hot flashes. MIP-1ß may be induced in response to an increase in IL-8 in women with hot flashes.

In conclusion, serum IL-8 concentrations in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women with hot flashes were significantly higher those in women without hot flashes, suggesting that IL-8 may be associated with peripheral vasodilation in women with hot flashes.

	Footnotes

 
First Published Online October 3, 2006

Abbreviations: HPA, Hypothalamic-pituitary-adrenal; MIP-1ß, macrophage inflammatory protein-1ß.

Received May 22, 2006.

Accepted September 26, 2006.

	References

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