JOURNAL OF BONE AND MINERAL RESEARCH Volume 5, Number 9, 1990 Mary Ann Liebert, Inc., Publishers
Estrogen Inhibits Release of Tumor Necrosis Factor from Peripheral Blood Mononuclear Cells in Postmenopausal Women STUART H. RALSTON,',' R. GRAHAM G. RUSSELL,' and MAXINE GOWEN'.3
ABSTRACT Cytokines, such as interleukin-1 (IL-1) and tumor necrosis factors (TNF), produced by cells of the monocyte-macrophage lineage in the local bone microenvironment, are potentially important local regulators of bone turnover. To investigate whether the protective effects of estrogen against postmenopausal bone loss may be mediated by inhibition of cytokine release, we studied the effects of 170-estradio1, dihydrotestosterone, and hydrocortisone on TNF release from human peripheral blood mononuclear cells (PBMC) in vitro. In unstimulated cells derived from eight postmenopausal women, seven of whom had osteoporotic vertebral fractures, 170-estradiol inhibited TNF release in a dose-dependent manner between and lo-'' M but had no consistent effect on cells derived from men o r premenopausal women. Dihydrotestosterone in concentrations of up to M had no effect on TNF release in any patient group, whereas hydrocortisone at M was a potent inhibitor of TNF release in all groups. Since TNF is a potent stimulator of bone resorption, the inhibitory effect of estrogen on TNF release may be part of the mechanism by which it exerts a protective effect on the skeleton in postmenopausal women. These observations may also be relevant in other inflammatory diseases of connective tissue, such as rheumatoid arthritis, in which disease activity may fluctuate as estrogen levels change - during the menstrual cycle, in pregnancy, and after the menopause.
INTRODUCTION after the menopause is the major cause of postmenopausal bone loss, and this loss can be prevented by estrogen replacement.('-31Until recently, the actions of estrogen on bone were thought to be indirect and mediated by changes in systemic calcium regulating hormones, such as 1,25-dihydroxyvitamin D,(4)and calcit ~ n i n . (Estrogen ~) receptors have now been shown to be present on osteoblastlike but the stimulatory effects of estrogen on these cells in vitro are only seen using supraphysiologic concentrations of estrogen. (') Furthermore, it has become apparent that bone formation and re-
E
STROGEN DEFICIENCY
sorption can be regulated by the many cytokines and growth factors that can be produced by bone and by immune cells within the local environment of bone.(lo) It is therefore possible that, in addition to these effects, estrogen may also act by altering the production of these locally produced mediators. For example, estrogens have recently been shown to enhance the production of insulinlike growth factor 1 (IGF-I) by bone cells.'") Other important cytokine mediators are interleukin-1 (IL-1), tumor necrosis factor (Y TNF-CY(cachetin), and TNF-0 (lymphotoxin). The principal source of IL-1 and TNF-(Yare cells of the monocyte-macrophage lineage, but these factors may also be produced by bone TNF-/3 however, is mainly de-
'Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Rd, Sheffield, S10 2RX, England.
'Present address: Rheumatic Diseases Unit, Northern General Hospital, Ferry Rd, Edinburgh, EH5 4DQ, Scotland. 'Present address: Bath Institute for Rheumatic Diseases, Trim Bridge, Bath, BAI 1 HD, England.
983
RALSTON ET AL.
984
rived from lymphocyte^.'^^^") All three cytokines are potent stimulators of bone resorption and modulators of osteoblast proliferation and function.‘”-”) Recently, Pacifici and his colleagues‘Is) demonstrated that cultured peripheral blood mononuclear cells from a subgroup of patients with osteoporosis released elevated amounts of IL-1 and that this could be suppressed to control levels by estrogen or protestagen hormone replacement therapy. Prompted by these observations, we studied the effects of sex hormones on the release of TNF by cultured peripheral blood mononuclear cells derived from normal healthy subjects and postmenopausal women.
MATERIALS AND METHODS Venous blood samples were obtained from four groups of subjects: eight healthy young men aged 24-32; eight healthy premenopausal women aged 22-34; eight healthy men aged 55-78 with no history of osteoporosis or other disorders of calcium metabolism; and eight postmenopausal women aged 52-75, one of whom was healthy and seven of whom had osteoporosis with vertebral compression fractures. Of the premenopausal women, three were taking estrogen- and progestagen-containing oral contraceptives, but no other subjects were taking corticosteroids, sex hormones, or other medications, such as nonsteroidal anti-inflammatory drugs, which may have affected cytokine release. Similarly, none of the patients were suffering from intercurrent illnesses at the time of the study that may have altered the release of cytokines from their peripheral blood cells. Peripheral blood mononuclear cells (PBMC) were isolated from a 10 ml heparinized blood sample by FicollHypaque density gradient centrifugation (Sigma, St. Louis, MO) and washed three times in phenol red-free RPMI-1640 medium supplemented with 10,OOO U/ml of penicillin, 10,OOO pg/ml of streptomycin, and 5% heat-inactivated bovine fetal calf serum that had been treated with charcoal to remove small molecules, such as steroid hormones. The estradiol concentration in the culture medium was undetectable using a radioimmunoassay with a lower limit of detection of 40 pmol/liter. All hormones (Sigma, St. Louis, MO) were initially dissolved in ethanol and subsequently in RPMI-1640 to give a final ethanol concentration of less than 1To. Lipopolysaccharide (LPS, Sigma, Poole, UK) was dissolved in RPMI-1640 to give a final concentration of 500 ng/ml. After separation and washing, PBMC were suspended in culture medium at a density of 1 x lo6 per ml and seeded at 1 x lo5 cells per well into round-bottomed %-well plates (Costar, Cambridge, MA). The cells were incubated at 37°C in air and 5% CO, for 24 h, and the supernatants carefully removed for TNF bioassay. TNF was measured using the TNF-sensitive cell line WEHI 164, clone 13, as described by Espevik and NissenMeyer.[zo)Briefly, WEHI cells were seeded into flat-bottomed 96-well microtiter plates in RPMI-1640 medium suplemented by a 1Vo penicillin-streptomycin solution (l0,OOO U/ml and 10,OOO pg/ml, respectively, GIBCO,
Paisley, UK) and 10% heat-inactivated fetal bovine serum (Northern Biological, Northumberland, UK) at a density of 2 x 104 cells per well and left for 4 h at 37°C in air and 5 % CO, to adhere. Samples for assay or human recombinant TNF-a standards (rhTNF), kindly donated by Dr. G.A. Adolf, Boeringer Ingelheim, were added to the cells, which were cultured for a further 24 h. The tertazolium salt 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 10 pl, Sigma; 5 mg/ml in phosphate-buffered saline) was added for the final 4 h of culture. The supernatant was aspirated from each well and the residue dissolved in isopropanol and 0.04 M hydrochloric acid. The plates were read spectrophotometrically on a microplate reader (Lab systems MC340) using 570 nm as test and 630 nm as reference wavelengths. Cell survival was calculated as the optical density of the sample well divided by the optical density of the control well multiplied by 100%. In our hands this assay had a lower detection limit of 0.001 units TNF per ml, with a working range of 0.001-1ooO u/ml. The intra-assay coefficient of variation over the working range was less than 13.5%. TNF (1 unit/ml) as measured in the assay was defined as that which stimulates half-maximal cell death in the murine L-M assay, in which human recombinant TNF has a specific activity of 6 x lo7 units/mg. The TNF concentration in the PBMC supernatants in each experiment was derived after appropriate dilution by comparison with a standard curve using rhTNFa. Using this assay system, hydrocortisone, 17P-estradio1, and dihydrotestosterone had no direct cytotoxic effect on the WEHI cells at concentrations of up to M (data not shown).
RESULTS In unstimulated cells from postmenopausal women, 17Pestradiol at concentrations ranging from lowL2 to M significantly inhibited TNF release in four experiments (representative experiment, Fig. 1) and caused a nonsignificant inhibition of TNF release over the same dose range in the remaining four (Table 1). When data were pooled and expressed as percentage inhibition from control values, 17P-estradiol inhibited TNF release in samples derived from postmenopausal women over the dose range lo-”M (Fig. 2). Dihydrotestosterone at a concentration of lo-* M had no inhibitory effect on TNF release in these patients (Table 2) or at lower concentrations of between and M (data not shown). In PBMCs derived from males and premenopausal women, neither 17p-estradiol nor dihydrotestosterone significantly altered TNF release at M (Table 2) or at lower concentrations of between M (data not shown). There was no signifiand cant difference between levels of basal or LPS-stimulated TNF release or in the response to any of the hormones in the three premenopausal women who were taking oral contraceptives (data not shown). Neither 170-estradiol nor dihydrotestosterone at concentrations of M altered TNF release from LPS-stimulated PBMCs in any patient group (Table 3), and there was similarly no effect at the lower hormone concentrations (data not shown). Hydrocortisone
985
ESTROGEN AND TNF at concentrations of M, however, consistently inhibited TNF release from unstimulated and LPS-stimulated PBMCs in every experiment from every patient group (Tables 2 and 3). The absolute amounts of TNF release by unstimulated cells varied markedly within patient groups (Table 4). Between-group comparisons showed that PBMCs from older males released less TNF than in the other groups, although the differences were statistically significant only with respect to premenopausal women (unstimulated PBMCs-) and postmenopausal women (LPS-stimulated PBMCs). Given the large interpatient variation in TNF levels, however, this finding is difficult to interpret. The incremental rise in TNF release with LPS stimulation was similar in all four patient groups (Table 4). There was no significant
12
10
IT T C
-14
10
correlation between the amounts of TNF released by unstimulated PBMCs and age ( r = 0.12, NS) or the amount of TNF released and the time since menopause in the postmenopausal group ( r = 0.18, NS). Experiments using PBMC from three patients (one male, one premenopausal female, and one postmenopausal female) established that the cytotoxicity mediated by PBMC supernatants (both unstimulated and LPS stimulated) was virtually abolished (> 99% inhibition) by a specific monoclonal antibody sufficient to neutralize 500 U/ml of TNF-cx (kindly donated by Adolf, Ernst Boeringer Institute fur Arneimittel Forshrung, Wien, Austria). This confirms that TNF was responsible for the cytotoxicity observed and suggests, moreover, that the main TNF species released by the PBMC in vitro was TNF-cx(cachectin), presumably derived from monocytes, rather than TNF-fi (lymphotoxin), derived from lymphocytes.
li
DISCUSSION 1c
-12
-8
-10
10
10
17-0 wstradlol
10
-6
10
(M)
-6
-6
10
10
DHT
HC
(M)
FIG. 1. Effect of 170-estradiol on release of TNF from PBMCs from a postmenopausal woman with osteoporosis. Points are means of quadruplicate determinations, bars are SEM. (*)p < 0.05 from control; (**)p < 0.01 from control; (***), < 0.001 fron control.
Tumor necrosis factors, like interleukin-1, are pleiotropic cytokines produced predominantly by cells of the monocyte-macrophage lineage but also by a variety of other cell types,‘”’ including those derived from human bone explants. ( 8 , 9 )Currently, both groups of cytokines are thought to play an important role in the pathogenesis of bone and cartilage destruction in such inflammatory conditions as rheumatoid arthritis(Z1.”land in osteolytic lesions associated with certain malignant diseases, such as myeloma.‘13~*31 Recent studies indicate that excessive IL-1 release may also be involved in the pathogenesis of osteop o r o s i ~ ( ’and ~~~ that ~ ) ovarian steroids may play a part in and its messenger RNA regulating the release of IL-l(19.24) from peripheral blood mononuclear cells. ( 2 5 1 An intriguing finding in this study was that 17&estradiol inhibited TNF release by unstimulated PBMC derived from eight postmenopausal women, seven of whom had
OF TNF RELEASED BY UNTREATED AND ESTROGEN-TREATED TABLE1. LEVELS PBMCs DERIVED FROM POSTMENOPAUSAL WOMEN
Patient
1 2 3 4b 5
6 1 8
Age (years)
Menopausaf age (Years)
Control culture (U/ml TNF)
75 70 68 54 55 70 72 14
28 24 23 6 10 21 24 22
11.4 (1.2) 132.0 (10.0) 9.2 (1.0) 23.5 (2.5) 49.0 (3.5) 1.10 (0.1) 144.0 (10) 23.5 (3.0)
ap < 0.05 from control cultures. bNonosteoporotic patient. cp < 0.01 from control cultures. dp < 0.02 from control cultures.
Estradioltreated M) culture (U/mf TNF)
7.4 104.0 4.9 16.4 25.0 0.53 125 17.6
(1.0)a (12.0) (1.0)a (3.0) (5.O)c
(0.1O)d (6.0) (2.0)
986
RALSTON ET AL.
osteoporotic fractures, but not by PBMC derived from premenopausal women or men. We did not perform experiments with the biologically less active isomer 17a-estradiol to confirm that this effect was indeed due to a specific interaction of the hormone with the estrogen receptor. Although possible, a nonspecific effect of high estrogen concentrations on the glucocorticoid receptor seems unlikely: first, the inhibitory effect of 17P-estradiol occurred at concentrations several orders of magnitude lower than those at which such an effect would be expected; second, the inhibitory effect was restricted to the postmenopausal patients; and third, no inhibitory effect on TNF release occurred with dihydrotestosterone at concentrations of up to
-e-*
1204.4
2
E 100-
8 E E
L
p
Estrogen Inhibits Release of Tumor Necrosis Factor from Peripheral Blood Mononuclear Cells in Postmenopausal Women STUART H. RALSTON,',' R. GRAHAM G. RUSSELL,' and MAXINE GOWEN'.3
ABSTRACT Cytokines, such as interleukin-1 (IL-1) and tumor necrosis factors (TNF), produced by cells of the monocyte-macrophage lineage in the local bone microenvironment, are potentially important local regulators of bone turnover. To investigate whether the protective effects of estrogen against postmenopausal bone loss may be mediated by inhibition of cytokine release, we studied the effects of 170-estradio1, dihydrotestosterone, and hydrocortisone on TNF release from human peripheral blood mononuclear cells (PBMC) in vitro. In unstimulated cells derived from eight postmenopausal women, seven of whom had osteoporotic vertebral fractures, 170-estradiol inhibited TNF release in a dose-dependent manner between and lo-'' M but had no consistent effect on cells derived from men o r premenopausal women. Dihydrotestosterone in concentrations of up to M had no effect on TNF release in any patient group, whereas hydrocortisone at M was a potent inhibitor of TNF release in all groups. Since TNF is a potent stimulator of bone resorption, the inhibitory effect of estrogen on TNF release may be part of the mechanism by which it exerts a protective effect on the skeleton in postmenopausal women. These observations may also be relevant in other inflammatory diseases of connective tissue, such as rheumatoid arthritis, in which disease activity may fluctuate as estrogen levels change - during the menstrual cycle, in pregnancy, and after the menopause.
INTRODUCTION after the menopause is the major cause of postmenopausal bone loss, and this loss can be prevented by estrogen replacement.('-31Until recently, the actions of estrogen on bone were thought to be indirect and mediated by changes in systemic calcium regulating hormones, such as 1,25-dihydroxyvitamin D,(4)and calcit ~ n i n . (Estrogen ~) receptors have now been shown to be present on osteoblastlike but the stimulatory effects of estrogen on these cells in vitro are only seen using supraphysiologic concentrations of estrogen. (') Furthermore, it has become apparent that bone formation and re-
E
STROGEN DEFICIENCY
sorption can be regulated by the many cytokines and growth factors that can be produced by bone and by immune cells within the local environment of bone.(lo) It is therefore possible that, in addition to these effects, estrogen may also act by altering the production of these locally produced mediators. For example, estrogens have recently been shown to enhance the production of insulinlike growth factor 1 (IGF-I) by bone cells.'") Other important cytokine mediators are interleukin-1 (IL-1), tumor necrosis factor (Y TNF-CY(cachetin), and TNF-0 (lymphotoxin). The principal source of IL-1 and TNF-(Yare cells of the monocyte-macrophage lineage, but these factors may also be produced by bone TNF-/3 however, is mainly de-
'Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Rd, Sheffield, S10 2RX, England.
'Present address: Rheumatic Diseases Unit, Northern General Hospital, Ferry Rd, Edinburgh, EH5 4DQ, Scotland. 'Present address: Bath Institute for Rheumatic Diseases, Trim Bridge, Bath, BAI 1 HD, England.
983
RALSTON ET AL.
984
rived from lymphocyte^.'^^^") All three cytokines are potent stimulators of bone resorption and modulators of osteoblast proliferation and function.‘”-”) Recently, Pacifici and his colleagues‘Is) demonstrated that cultured peripheral blood mononuclear cells from a subgroup of patients with osteoporosis released elevated amounts of IL-1 and that this could be suppressed to control levels by estrogen or protestagen hormone replacement therapy. Prompted by these observations, we studied the effects of sex hormones on the release of TNF by cultured peripheral blood mononuclear cells derived from normal healthy subjects and postmenopausal women.
MATERIALS AND METHODS Venous blood samples were obtained from four groups of subjects: eight healthy young men aged 24-32; eight healthy premenopausal women aged 22-34; eight healthy men aged 55-78 with no history of osteoporosis or other disorders of calcium metabolism; and eight postmenopausal women aged 52-75, one of whom was healthy and seven of whom had osteoporosis with vertebral compression fractures. Of the premenopausal women, three were taking estrogen- and progestagen-containing oral contraceptives, but no other subjects were taking corticosteroids, sex hormones, or other medications, such as nonsteroidal anti-inflammatory drugs, which may have affected cytokine release. Similarly, none of the patients were suffering from intercurrent illnesses at the time of the study that may have altered the release of cytokines from their peripheral blood cells. Peripheral blood mononuclear cells (PBMC) were isolated from a 10 ml heparinized blood sample by FicollHypaque density gradient centrifugation (Sigma, St. Louis, MO) and washed three times in phenol red-free RPMI-1640 medium supplemented with 10,OOO U/ml of penicillin, 10,OOO pg/ml of streptomycin, and 5% heat-inactivated bovine fetal calf serum that had been treated with charcoal to remove small molecules, such as steroid hormones. The estradiol concentration in the culture medium was undetectable using a radioimmunoassay with a lower limit of detection of 40 pmol/liter. All hormones (Sigma, St. Louis, MO) were initially dissolved in ethanol and subsequently in RPMI-1640 to give a final ethanol concentration of less than 1To. Lipopolysaccharide (LPS, Sigma, Poole, UK) was dissolved in RPMI-1640 to give a final concentration of 500 ng/ml. After separation and washing, PBMC were suspended in culture medium at a density of 1 x lo6 per ml and seeded at 1 x lo5 cells per well into round-bottomed %-well plates (Costar, Cambridge, MA). The cells were incubated at 37°C in air and 5% CO, for 24 h, and the supernatants carefully removed for TNF bioassay. TNF was measured using the TNF-sensitive cell line WEHI 164, clone 13, as described by Espevik and NissenMeyer.[zo)Briefly, WEHI cells were seeded into flat-bottomed 96-well microtiter plates in RPMI-1640 medium suplemented by a 1Vo penicillin-streptomycin solution (l0,OOO U/ml and 10,OOO pg/ml, respectively, GIBCO,
Paisley, UK) and 10% heat-inactivated fetal bovine serum (Northern Biological, Northumberland, UK) at a density of 2 x 104 cells per well and left for 4 h at 37°C in air and 5 % CO, to adhere. Samples for assay or human recombinant TNF-a standards (rhTNF), kindly donated by Dr. G.A. Adolf, Boeringer Ingelheim, were added to the cells, which were cultured for a further 24 h. The tertazolium salt 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 10 pl, Sigma; 5 mg/ml in phosphate-buffered saline) was added for the final 4 h of culture. The supernatant was aspirated from each well and the residue dissolved in isopropanol and 0.04 M hydrochloric acid. The plates were read spectrophotometrically on a microplate reader (Lab systems MC340) using 570 nm as test and 630 nm as reference wavelengths. Cell survival was calculated as the optical density of the sample well divided by the optical density of the control well multiplied by 100%. In our hands this assay had a lower detection limit of 0.001 units TNF per ml, with a working range of 0.001-1ooO u/ml. The intra-assay coefficient of variation over the working range was less than 13.5%. TNF (1 unit/ml) as measured in the assay was defined as that which stimulates half-maximal cell death in the murine L-M assay, in which human recombinant TNF has a specific activity of 6 x lo7 units/mg. The TNF concentration in the PBMC supernatants in each experiment was derived after appropriate dilution by comparison with a standard curve using rhTNFa. Using this assay system, hydrocortisone, 17P-estradio1, and dihydrotestosterone had no direct cytotoxic effect on the WEHI cells at concentrations of up to M (data not shown).
RESULTS In unstimulated cells from postmenopausal women, 17Pestradiol at concentrations ranging from lowL2 to M significantly inhibited TNF release in four experiments (representative experiment, Fig. 1) and caused a nonsignificant inhibition of TNF release over the same dose range in the remaining four (Table 1). When data were pooled and expressed as percentage inhibition from control values, 17P-estradiol inhibited TNF release in samples derived from postmenopausal women over the dose range lo-”M (Fig. 2). Dihydrotestosterone at a concentration of lo-* M had no inhibitory effect on TNF release in these patients (Table 2) or at lower concentrations of between and M (data not shown). In PBMCs derived from males and premenopausal women, neither 17p-estradiol nor dihydrotestosterone significantly altered TNF release at M (Table 2) or at lower concentrations of between M (data not shown). There was no signifiand cant difference between levels of basal or LPS-stimulated TNF release or in the response to any of the hormones in the three premenopausal women who were taking oral contraceptives (data not shown). Neither 170-estradiol nor dihydrotestosterone at concentrations of M altered TNF release from LPS-stimulated PBMCs in any patient group (Table 3), and there was similarly no effect at the lower hormone concentrations (data not shown). Hydrocortisone
985
ESTROGEN AND TNF at concentrations of M, however, consistently inhibited TNF release from unstimulated and LPS-stimulated PBMCs in every experiment from every patient group (Tables 2 and 3). The absolute amounts of TNF release by unstimulated cells varied markedly within patient groups (Table 4). Between-group comparisons showed that PBMCs from older males released less TNF than in the other groups, although the differences were statistically significant only with respect to premenopausal women (unstimulated PBMCs-) and postmenopausal women (LPS-stimulated PBMCs). Given the large interpatient variation in TNF levels, however, this finding is difficult to interpret. The incremental rise in TNF release with LPS stimulation was similar in all four patient groups (Table 4). There was no significant
12
10
IT T C
-14
10
correlation between the amounts of TNF released by unstimulated PBMCs and age ( r = 0.12, NS) or the amount of TNF released and the time since menopause in the postmenopausal group ( r = 0.18, NS). Experiments using PBMC from three patients (one male, one premenopausal female, and one postmenopausal female) established that the cytotoxicity mediated by PBMC supernatants (both unstimulated and LPS stimulated) was virtually abolished (> 99% inhibition) by a specific monoclonal antibody sufficient to neutralize 500 U/ml of TNF-cx (kindly donated by Adolf, Ernst Boeringer Institute fur Arneimittel Forshrung, Wien, Austria). This confirms that TNF was responsible for the cytotoxicity observed and suggests, moreover, that the main TNF species released by the PBMC in vitro was TNF-cx(cachectin), presumably derived from monocytes, rather than TNF-fi (lymphotoxin), derived from lymphocytes.
li
DISCUSSION 1c
-12
-8
-10
10
10
17-0 wstradlol
10
-6
10
(M)
-6
-6
10
10
DHT
HC
(M)
FIG. 1. Effect of 170-estradiol on release of TNF from PBMCs from a postmenopausal woman with osteoporosis. Points are means of quadruplicate determinations, bars are SEM. (*)p < 0.05 from control; (**)p < 0.01 from control; (***), < 0.001 fron control.
Tumor necrosis factors, like interleukin-1, are pleiotropic cytokines produced predominantly by cells of the monocyte-macrophage lineage but also by a variety of other cell types,‘”’ including those derived from human bone explants. ( 8 , 9 )Currently, both groups of cytokines are thought to play an important role in the pathogenesis of bone and cartilage destruction in such inflammatory conditions as rheumatoid arthritis(Z1.”land in osteolytic lesions associated with certain malignant diseases, such as myeloma.‘13~*31 Recent studies indicate that excessive IL-1 release may also be involved in the pathogenesis of osteop o r o s i ~ ( ’and ~~~ that ~ ) ovarian steroids may play a part in and its messenger RNA regulating the release of IL-l(19.24) from peripheral blood mononuclear cells. ( 2 5 1 An intriguing finding in this study was that 17&estradiol inhibited TNF release by unstimulated PBMC derived from eight postmenopausal women, seven of whom had
OF TNF RELEASED BY UNTREATED AND ESTROGEN-TREATED TABLE1. LEVELS PBMCs DERIVED FROM POSTMENOPAUSAL WOMEN
Patient
1 2 3 4b 5
6 1 8
Age (years)
Menopausaf age (Years)
Control culture (U/ml TNF)
75 70 68 54 55 70 72 14
28 24 23 6 10 21 24 22
11.4 (1.2) 132.0 (10.0) 9.2 (1.0) 23.5 (2.5) 49.0 (3.5) 1.10 (0.1) 144.0 (10) 23.5 (3.0)
ap < 0.05 from control cultures. bNonosteoporotic patient. cp < 0.01 from control cultures. dp < 0.02 from control cultures.
Estradioltreated M) culture (U/mf TNF)
7.4 104.0 4.9 16.4 25.0 0.53 125 17.6
(1.0)a (12.0) (1.0)a (3.0) (5.O)c
(0.1O)d (6.0) (2.0)
986
RALSTON ET AL.
osteoporotic fractures, but not by PBMC derived from premenopausal women or men. We did not perform experiments with the biologically less active isomer 17a-estradiol to confirm that this effect was indeed due to a specific interaction of the hormone with the estrogen receptor. Although possible, a nonspecific effect of high estrogen concentrations on the glucocorticoid receptor seems unlikely: first, the inhibitory effect of 17P-estradiol occurred at concentrations several orders of magnitude lower than those at which such an effect would be expected; second, the inhibitory effect was restricted to the postmenopausal patients; and third, no inhibitory effect on TNF release occurred with dihydrotestosterone at concentrations of up to
-e-*
1204.4
2
E 100-
8 E E
L
p
