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性賀爾蒙對培養皮膚真皮微血管內皮細胞之影響 The Effect of Sex Steroid Hormones on the Activation of
Human Dermal Microvascular Endothelial Cells 計畫編號： NSC 89-2314-B-006-092 執行年限：88 年 8 月 1 日至 89 年 7 月 31 日
主持人：Yu-Yun Lee, MD 國立成功大學醫學院皮膚部
共同主持人：WenChieh Chen, MD 國立成功大學醫學院皮膚部 計畫參與人員： Wen-Chuan Hsieh, MSc 國立成功大學醫學院皮膚部 中文摘要
膠原病，如紅斑性狼瘡及進行性全身型 硬皮症在女性的發生率遠高於男性。愈來愈多 的證據顯示內皮細胞不正常活化可能扮演關鍵 性的角色。性賀爾蒙除了具有免疫調節的性質 之 外 ， 也 會 影 響 內 皮 細 胞 cellular adhesion molecules (CAMs)的表現。有些致發炎物質，
如 IL-1 與 TNF-α 對內皮細胞之 CAMs 有刺激 作用，使 ICAM-1、VCAM-1 與 E-selectin 表現 增加，而這些物質對於顆粒球或單核球細胞黏 著於內皮細胞的過程非常重要。目前有關性賀 爾蒙影響內皮細胞的研究大多是以人類臍帶靜 脈內皮細胞 (HUVEC)為主，但不同來源的內 皮細胞性質各不相同，本實驗利用人類真皮微 血管內皮細胞株(HMEC-1, CDC, USA 提供)，
生長於不含酚紅之培養液中，測定不同的性賀 爾蒙對經 IL-1 (50 U/ml)/TNF-α (50 U/ml)刺激 後之微血管內皮細胞上 CAMs 表現的影響，並 與 HUVEC 比較。在 HMEC-1 方面， ICAM-1 的表現受 dexamethasone 的抑制最為明顯、而 dihydrotestosterone 的抑制作用較 testosterone 強，但動情二醇並不影響 ICAM-1 的表現，可 能是因為此細胞株缺乏女性賀爾蒙受體。我們 的研究顯示：雖然動情二醇並不會促進內皮細 胞中經 IL-1/TNF-α所刺激之反應，但男性賀爾 蒙卻對此有抑制作用，並可能因此保護組織免 於發炎。由這些結果看來，男性賀爾蒙在 IL- 1/TNF-α媒介之反應中，具有保護皮膚免於發 炎的角色。這與紅斑性狼瘡及硬皮症好發於女 性，並以皮膚為主要侵犯器官的情形不謀而 合。
關鍵詞：性賀爾蒙、人類真皮微血管內皮細胞 Abstr act:
Connective tissue diseases, such as systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS), occur far more frequently in female than in male.
Accumulating evidences show that dysfunction or activation of endothelial cells plays a crucial role.
In addition to possessing immunoregulatory properties, sex steroids were shown to affect the expression of endothelial adhesion molecules.
Some proinflammatory cytokines such as IL-1 and TNF-α can upregulate some adhesion molecules
(CAMs) on the endothelial cell surface, like ICAM-1, VCAM-1 and E-selectin, which are important for adhesion of granulocytes and monocytes. Heterogeneity of vascular endothelial cells from different origins is well known, however, most current studies address the influence of sex hormones on human umbilical vein endothelial cells (HUVEC). To study the influence of some steroid hormones on the IL-1 (50 U/mL)/ TNF-α (50 U/mL) stimulated expression of CAMs on microvascular endothelial cells, we used HMEC-1 cells, a human dermal microvascular endothelial cell line (generously provided by CDC, Atlanta, USA) in phenol-red free culture medium, and did a parallel study on HUVEC. In HMEC-1 cells, Dexamethasone (DEX) was most potent in inhibiting the expression of ICAM-1, most pronounced at 24h.
Dihydrotestosterone (DHT) had stronger inhibitory effect than testosterone (T). Estrogen (estradiol; E2) showed no regulatory effect on the endothelial activation, probably due to the absence of estrogen receptor in this cell line. Our study showed that, while E2 did not appear to promote the pro-inflammatory effects of IL- 1/TNF-α on endothelial cells, androgens did show some suppressive effects, hence might serve to protect the target tissues from inflammation.
These findings, in particular the one on HDMEC, lend to the support of a protective role of androgens from the IL-1/TNF-α mediated inflammation in the skin, and are consistent with the strong female predilection in lupus erythematosus and scleroderma where the skin is one of the major organs affected.
Keywor ds: Steroid sex hormones, human dermal microvascular endothelial cells, activation markers
Backgr ound:
Connective tissue diseases, such as systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS), occur far more frequently in female than in male.1
2 Accumulating evidences show that dysfunction or activation of vascular endothelial cells plays a crucial role.2 Sex steroids were shown to affect the expression of endothelial adhesion molecules, in addition to the finding that estrogens were immunostimulatory with respect to humoral immunity and immunosuppressive with respect to cellular immunity, while androgens were generally immunosuppressive.3 The expression of cellular adhesion molecules (CAMs) on vascular endothelial cells determines the pattern of migration and extravasation of leucocytes in inflammation and immune reaction.4,5 Most published data of in vitro human endothelial cell studies used human umbilical vascular endothelial cells (HUVEC), however, it has become increasingly recognized that not all endothelial cells are alike.6 Androgen receptors as well as steroid 5α-reductase, the enzyme which converts testosterone (T) to the more potent dihydrotestosterone (DHT), have both been demonstrated to be present in human dermal microvascular endothelial cells (HDMEC).7-9 Pur pose:
To determine whether the sex steroid hormones (corticosteroid vs. androgen vs.
estrogen) modulate the expression of specific markers of activated HDMEC. A comparison to HUVEC is made.
Mater ials and Methods:
A transformed HDMEC cell line, HMEC-1, (generously offered by Dr. FC Candal, CDC, USA) and primary culture of HUVEC were used.
The cells were grown in phenol red-free EGM (Clonetics, USA) containing 2 % fetal bovine serum (FBS). Specific monoclonal antibodies (Dako, Denmark) were used to test the expression of the androgen and estrogen receptors (AR, ER), respectively.
We first studied the dynamic of cytokines’
effect on the expression of CAMs by HMEC- 1 up to 24 hours. IL-1 β and TNF-α (R&D, USA), at final concentration of 50 U/ml each, were used to stimulate the HMEC-1 cells cultured in 96-well plate (2×104 cells/well). The expression of ICAM- 1, VCAM-1 and E-selectin on the cellular surface was measured by ELISA (Dynatech, USA) using specific monoclonal antibodies (all from Serotec, USA).
At the second step, sex steroid hormones alone were given to the cultured cells to see if
they could influence the basal constitutive expression of CAMs.
Then we studied the effect of sex steroid hormones on regulating the cytokines stimulated expression of CAMs. Before the administration of cytokines and hormones to FBS-supplemented medium, the HMEC-1 cells were starved first by switching the medium from the FBS containing medium (2 %) to serum-free medium for 24 hours (starvation). Sex steroid hormones (10-6 to 10-8 M), including T, DHT, dexamethasone (DEX) and 17β-estradiol (E2) (all from Sigma, USA) were added to the culture medium simultaneously with the IL-1 β/TNF-α. After incubation for 2, 4, 6, 12, 18, 24h, the supernatant were removed. The expression of ICAM-1, VCAM-1 and E-selectin on the cellular surface was measured with ELISA.
A parallel study was done on HUVEC.
To test the presence of AR and ER on the cell lines, the cell lines were first cultured in serum-containing medium (2 % FBS), and then switched to serum-free media for 24 hours (starvation). T and E2 were then added to the cell lines (48 hr for HMEC-1 and 24 hr for HUVEC), respectively, to boost the expression of AR and ER, respectively. The expression of AR and ER on the endothelial cells was studied by immunocytochemistry, with testis and ovary used as positive controls, respectively.
Results:
The basal constitutive expression of ICAM- 1 was very low in HMEC as well as in HUVEC.
The basal expression of VCAM-1 and E-selectin were also very low in HUVEC, but only barely detectable in HMEC-1. The steroid hormones under examination, when given in the absence of cytokines, had no effect on the expression of CAMs.
In HMEC-1, DEX was most potent to inhibit the IL-1 (50 U/ml)/ TNF-α (50 U/ml) stimulated expression of ICAM-1. The effect was most pronounced at 24 hours of treatment. DHT had stronger inhibitory effect than T. E2 was found to exert no regulatory effect on the IL- 1/TNF-α stimulated endothelial expresion of CAMs, probably due to the absence of estrogen receptor (ER) in this cell line as determined by immunostaining. The expression of VCAM-1 and E-selectin was beyond the lower limitation of our detection system, even after the stimulation of the added IL-1/TNF-α. ER and AR were not demonstrated in HMEC-1 cells, even after the treatment of 10-6 M of E2 and testosterone for 48
3 hr, respectively.
By contrast, in HUVEC, DEX inhibited the IL-1 (50 U/ml)/TNF-α (50 U/ml) stimulated expression of E-selectin (at 4h), VCAM (at 6h) and ICAM-1 (beginning at 18h). DHT and T inhibited the expression of VCAM-1 (at 6 hr) but not E-selectin, and there was no difference between their inhibitory effect. Immunostaining of the HUVEC revealed very weak reaction of ER on the cells, even after treating with 10-6 M E2 for 24 hr. No AR was detectable in HUVEC.
Discussion:
Variable kinetics of appearance of E- selectin, VCAM-1 and ICAM-1 has been shown in different experimental models of inflammation.10 One factor to be considered is to give cytokines and hormones simultaneously at time 0 or sequentially. Moreover, the assessment of hormone responses in vitro is complicated by incomplete definition of critical culture conditions.11-14 To address whether the observed inhibition was due to a receptor-mediated or nonspecific steroid effect, we tested the expression of ER and AR. In this study, sex hormones was added simultaneously with cytokines (IL-1 and TNF-α) to cultured cells, and FBS and phenol red-free medium was used to exclude any basal estrogen/androgen effect and artifactual ER binding, respectively.
The detection of ERs on blood vessels has been variable, dependent upon the vascular bed and the technique used. In one study14, HUVEC pretreated with high-dose E2 (1000 ng/ml, about 0.5
× 10
-6M)) for 3 and 24 hr was shown to be ER-negative, and the induction of ER message was first observed after 48 h E2 pretreatment. In our study, ER and AR were not demonstrated in HMEC-1 cells, even after the treatment of 10-6 M of E2 and testosterone for 48 hr, respectively. The very weak ER expression in HUVEC may be due to the insufficient duration of treatment (24 hr) in our experiment.There is little information about hormonal regulation of CAMs expression on HDMEC. As for HUVEC, controversial results have been reported regarding the enhanced or reduced induction of CAMs by estradiol.11-14 Our study showed, in HMEC-1 as well as in HUVEC, there was basal expression of ICAM-1, while the expression of E-selectin and VCAM-1 was only became detectable after stimulation by pro- inflammatory cytokines (IL-1 + TNF-α).
Maximal expression of E-selectin in vitro
occurred 4 hours after activation and declined thereafter. VCAM-1 appeared at slightly later times than E-selectin, with onset in 6-12 hours.
The expression of ICAM-1 was strongly upregulated by cytokine activation, and the maximal effect was observed at 18-24 hr. The time course of expression of these CAMs was compatible to that in previous reports.15,16
The steroid hormones under examination, when given in the absence of cytokines, had no impact on the regulation of CAMs. Co- administered with cytokines, DEX inhibited the expressions of E-selectin, VCAM-1 and ICAM-1 in HUVEC, but it inhibited only ICAM-1 expression in HMEC-1. DHT was stronger than T in inhibiting the cytokine-activated expression of ICAM-1 in HMEC-1. They had similar potency to inhibit the VCAM-1 expression in HUVEC. E2 did not show regulating effect on the expression of CAMs in HMEC-1, nor in HUVEC.
Our study showed that while E2 did not appear to promote the pro-inflammatory effects of IL-1/TNF-α on endothelial cells, androgen hormones did show some suppressive effects, hence might serve to protect the target tissues from inflammation. These findigs, in particular the one on HDMEC, lend to the support a protective role of androgens on the IL-1/TNF-α mediated inflammation in the skin, and are consistent with the strong female predilection in lupus erythematosus and scleroderma where the skin is one of the major organs affected. Since AR was absent in both cell types, further study is needed to explore the inhibitory effect of androgens on the expression of CAMs.
Self Assessment:
At the beginning of our experiment, it took us a lot of time to find out the most suitable culture medium for the HMEC-1, which requires special media and conditions. It was difficult to remove the steroid hormones contained in FBS to exclude their basal stimulatory effect on the cultured cells and at the same time to maintain the cell viability and optimal cell growth. During the course of the study, we became realized that we had to run a parallel study on HUVEC for comparison of the effects under study and also had to find out whether there was expression of AR and ER and how to best induce their expression. We were fortunate enough to have a lot of valuable advice from Dr. L-Y. Chen, professor from the Department of Physiology, NCKU, who has extensive experience in studying
4 sex hormones on HUVEC. More work need to be done to define the optimal expression of sex hormone receptors (AR versus ER) and its correlation to their inhibitory effect on the cytokine-activated expression of CAMs. It would be also interesting to do a dose-response study of the cytokine activation. In sum, the present study helped us to establish the basics in our lab for further study of the effects of sex hormones on endothelial cells. The results also support a protective role of androgens in males from inflammatory disease such as lupus erythematous and scleroderma.
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