Elsevier

Bone

Volume 29, Issue 1, July 2001, Pages 30-34
Bone

The effect of 17β-estradiol on production of cytokines in cultures of peripheral blood

https://doi.org/10.1016/S8756-3282(01)00468-9Get rights and content

Abstract

Estrogen’s action on bone may be mediated by cytokines produced by monocytes. We have reported a decreased ratio of interleukin-1β (IL-1β) to interleukin-1 receptor antagonist (IL-1ra) produced by whole blood cultures in vivo in women taking hormone replacement therapy (HRT). Also, one study has shown an effect of estradiol on tumor necrosis factor-α (TNF-α) secretion by separated monocytes in vitro. The aim of this study was to evaluate the effect of estrogen in vitro on the secretion of cytokines using whole blood cultures. Subjects consisted of 12 healthy postmenopausal women, ages 57–69 years, 4–20 years since menopause. Cytokines IL-1β, interleukin-1α (IL-1α), IL-1ra, interleukin-6 (IL-6), TNF-α, and granulocyte macrophage-colony stimulating factor (GM-CSF) were measured in unstimulated and in stimulated (500 ng/mL lipopolysaccharide [LPS]) whole blood cultures treated with 17β-estradiol (E2) at concentrations of 10−12–10−6 mol/L. We found significant decreases in the spontaneous secretion of IL-6, TNF-α, IL-1ra, IL-1β, and ratio of IL-1β/IL-1ra compared with control, at physiological concentrations of E2. The action of E2 was blocked by the use of the antiestrogen ICI 182780 in coculture. A decrease in cytokine secretion was not observed when the inactive form of estrogen, 17α-estradiol, was used in place of 17β-estradiol. GM-CSF and IL-1α were not detectable in unstimulated cultures. Cytokine levels measured in stimulated cultures were not attenuated by treatment with E2. We conclude that E2 inhibits the spontaneous secretion of cytokines measured in whole blood cultures at physiological concentrations, and that the powerful stimulatory effect of LPS prevents any significant inhibition by E2 in stimulated cultures.

Introduction

The proinflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), IL-6, and granulocyte macrophage-colony stimulating factor (GM-CSF) stimulate the proliferation and differentiation of osteoclasts leading to an increase in bone resorption and subsequent bone loss. These cytokines are produced mainly by mononuclear cells. One of the ways in which estrogen may prevent bone resorption is by modulating the secretion of cytokines produced by bone marrow monocytes. It is known that estrogen receptors are present in mononuclear cells11 and peripheral mononuclear cells have been used as a model for bone marrow monocytes.1 Previous studies on the effect of estrogen in vitro on peripheral blood mononuclear cells have been inconclusive.

17β-Estradiol has been shown to attenuate the secretion of TNF-α in separated mononuclear cells taken from eight postmenopausal women, seven of whom were osteoporotic (at least one vertebral fracture).8 The inhibitory effect observed on TNF-α secretion was not significant in four of the eight women, however, and the effect was not seen in premenopausal women or men.

A study of 11 normal postmenopausal women regarding the effect of estrogen on the secretion of IL-1 by mononuclear cells showed no significant attenuation of IL-1 production by these cells.10 In fact, nonsignificant increases in IL-1 bioactivity were observed at doses of 10−10–10−7 mol/L 17β-estradiol.

Polan et al. described significant suppression of IL-1β mRNA levels by 17β-estradiol at doses of 10−7–10−5 mol/L using steady-state IL-1β mRNA production as a measure of IL-1β activity. However, subjects for this study were healthy premenopausal women and samples were obtained during the luteal phase of the menstrual cycle.6

In a previous publication we evaluated different methods of measuring cytokines in the peripheral circulation. A method of measuring secreted cytokines using stimulated whole blood cultures was judged to be a suitable alternative to the measurement of cytokines expressed by separated peripheral blood mononuclear cells (PBMCs).9 The whole blood cultures responded to stimulation by LPS and hydrocortisone in a similar way to the separated PBMCs. In addition, we showed that the ratio IL-1β/IL-1ra (a measure of IL-1β activity) was lower in estrogen-treated postmenopausal women compared with controls. In this study we assess the response of whole blood cytokine secretion to 17β-estradiol in vitro.

The objectives of this study were: (a) to determine the dose effect of 17β-estradiol on spontaneous and stimulated secretion of cytokines measured in whole blood cultures taken from healthy postmenopausal women; and (b) to evaluate the specificity of this response by the use of an estrogen antagonist (ICI 182780) or the inactive form of estrogen (17α-estradiol).

Section snippets

Study subjects

Subjects included 12 healthy postmenopausal women, ages 57–69 years, 4–20 years since menopause. The women were either relatives of staff working in the Osteoporosis Centre, Northern General Hospital, Sheffield, or women recruited from local general practitioners. These women had no evidence of osteoporosis (i.e., a lumbar spine bone density T score >2.5 and an absence of vertebral fracture). The exclusion criteria for this study were: the presence of inflammatory diseases; diseases known to

Response of spontaneous production of cytokines to 17β-estradiol

Levels of secreted IL-1ra were decreased by 43% (±16% CI) at 10−12 mol/L and by 37% (±21% CI) at 10−10 mol/L 17β-estradiol compared with control (p′ < 0.01, p′ < 0.05, respectively) (Figure 1A). IL-1β decreased by 94% (±19% CI) at 10−10 mol/L 17β-estradiol (p′ < 0.05) and by 96% (±17%) at 10−12 mol/L (p′ < 0.05) (Figure 1D). The ratio IL-1β/IL-1ra was decreased significantly by 93% (±20% CI) at 10−10 mol/L and by 93% (±18% CI) at 10−8 mol/L (p′ < 0.05) (Figure 1D, E). There was a significant

Discussion

In this study we have shown that 17β-estradiol inhibits the spontaneous secretion of IL-1β, IL-1ra, IL-1β/IL-1ra, IL-6, and TNF-α measured in whole blood cultures at physiological concentrations. We also demonstrated the specificity of this response by using an antiestrogen (ICI 182780) and the inactive form of estrogen, 17α-estradiol. LPS-stimulated secretion of cytokines, however, was not attenuated by estradiol.

It is estimated that a dose of 10−10 mol/L 17β-estradiol would be equivalent to

Acknowledgements

The authors thank Dr. J. A. Clowes for her contribution in recruiting the subjects for this study.

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