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趨化激素接受體於人類真皮微血管內皮細胞之表現
Chemokine receptor expression on human dermal microvascular endothelial cells 計畫編號： NSC 89-2314-B-006-186
執行年限：90 年 8 月 1 日至 91 年 7 月 31 日
主持人：陳文杰主治醫師 國立成功大學醫學院皮膚科 共同主持人：王崇任醫師 國立成功大學醫學院風濕免疫科
計畫參與人員：楊倍昌教授、張雅南研究生 國立成功大學醫學院微免所
中文摘要
血管內皮細胞的活化在發炎反應的啟動居關鍵 性的角色。吾人之前的研究顯示雄性激素，特別 是雙氫睪固酮，可以抑制皮膚血管內皮細胞 (HMEC-1 細 胞 株 ) 經 IL-1/TNF- 活 化 後 ICAM-1 的表現。本實驗中吾人進一步檢查趨化 激素在 HMEC-1 的表現以及其是否受到類固 醇，包括性賀爾蒙，與 genistein 的調控。GRO-, IL-8, MCP-1 等三種趨化激素在 HMEC-1 的表現 很低，而在 HUVEC 的表現較強，分別約是 30、
6.5 及 26 倍強。對於刺激趨化激素在 HUVEC 的表現，IL-1 (100 ng)比 TNF- (100 ng)強。
HMEC-1 細胞經 IL-1 (100 ng)/TNF- (100 ng) 刺激 48 小時後，只有 IL-8 的表現增加，此一活 化可被 10-6M 的 dexamethasone 些微的抑制，而 性賀爾蒙與 genistein 並無抑制的作用。
關鍵詞: 真皮血管內皮細胞，炎症，趨化激素，
性賀爾蒙，類固醇， genistein
Abstract
Activation of endothelium is a critical event during the initiation of inflammatory processes. In our previous study, we showed that androgens, especially the dihydrotestosterone, could down-regulated the expression of ICAM-1 on IL-1/TNF- activated HMEC-1 cells. In this experiment, we further examined the expression of various chemokines and their regulation by steroids, including sex hormones, and genistein.
The basal constitutive expression of GRO-, IL-8, and MCP-1 was very low in HMEC-1 but higher in HUVEC (30-, 6.5-, 26-folds, respectively).
IL-1 (100 ng) was stronger than TNF- (100 ng) to stimulate the expression of the chemokines in HUVEC. In HMEC-1, only the expression of IL-8, but not GRO- and MCP-1 could be enhanced by treatment with IL-1/TNF- for 48 h. While 10-6M dexamethasone had mild inhibitory effect
on IL-8 expression, steroid hormones (testosterone, dihydrotestosterone and 17-estradiol) and genistein showed no regulatory effect.
Keywords: dermal microvascular endothelial cells, inflammation, chemokines, sex steroids,
glucocorticoids, genistein
Background
Most of the connective tissue diseases predominate in women [1]. One of the cardinal features of connective tissue diseases is vasculitis or vasculopathy involving small blood vessels.
The exact mechanisms by which sex hormones modulate disease activity are incompletely understood [2,3]. Injury or dysfunction of the dermal microvascular endothelial cells with activated expression of certain cell adhesion molecules (CAMs) was suggested to be the primary pathogenic events [4,5]. Leukocyte recruitment to sites of inflammation involves a multi-step process mediated by a series of sequential interactions of endothelial cells with various inflammatory cells and concerted expression of various combinations of selectins, integrins, and chemoattractants [6,7]. Sex steroids were shown to affect the endothelial expression of CAMs and possess immunoregulatory properties [8]. In our previous study, we showed androgens, especially dihydrotestosterone (DHT) had small, but statistically significant suppressive effect in HMEC-1 only, while estrogen exhibited no regulatory function in HMEC-1 as well as in human umbilical vein endothelial cells (HUVEC).
No obvious expression of estrogen and androgen receptors could be demonstrated in both cells by immunostaining [9].
Chemokines are a superfamily of 8-10 kDa basic proteins that are expressed and secreted by many cell types, including leukocytes and endothelial cells [10]. At least 40 identified members of this family can be divided into 4 subclasses termed
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CXC, CC, C (Lymphotactin) and CX3C (Fractalkine) based on structure and chromosomal localization [9]. Previous study showed that upon stimulation with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), both HDMECs and HMEC-1 expressed high levels of IL-8, GRO-, and monocyte chemoattractant protein-1 (MCP-1) [11]. This study was aimed to observe the effect of dexamethasone (DEX), testosterone (T), 5-dihydrotestosterone (DHT),17-estradiol (E2) and genistein (G) on the in vitro expression of IL-8, GRO-, and MCP-1 in human dermal microvascular endothelial cells.Materials and Methods
Cells from a transformed human dermal microvascular endothelial cell line, HMEC-1, (generously offered by Dr. FC Candal, CDC, USA) and primary culture of HUVEC were used. The cells were cultured in phenol red-free culture medium (EGM, Clonetics, MD, USA) containing 2 % fetal bovine serum (FBS).
The dynamic of cytokines’ effect on the expression of chemokines by HMEC- 1 up to 48 hours (0,6,12,24,48 h) was examined first. TNF-
and IL-1 (RD, MN, USA), at final concentration of 1000 ng/ml and 1000 U/ml, respectively, were used to stimulate the cultured HMEC-1 cells. The expression of IL-8, GRO-, and MCP-1 in the supernatant was measured by ELISA in triplicate (Dynatech, PA, USA) using specific monoclonal antibodies (all from R & D,).
At the second step, the individual steroid hormones (T, DHT, DEX and E2) and G, each at 10-6 M, were applied to the cell cultures to see if they could influence the endothelial basal constitutive expression of chemokines. Then, the tested agents and IL-1/TNF- at the aforementioned concentration were simultaneously added to the culture medium for 48 h to examine the amount of released chemokines in supernatant.
No cytokines and hormones were added in controls.
As for statistical analysis, values of ELISA studies represented the mean ± SE of separate determination in six different wells from three different experiments. Student’s t test was used for comparison of the means. Differences of p < 0.05 were considered significant.
Results
The basal constitutive expression of GRO-, IL-8, and MCP-1 was very low in HMEC-1, being 5594 111 pg/ml, 1255 35 pg/ml and 276 0 pg/ml, respectively. Higher basal level was found in HUVEC, with GRO-, IL-8, and MCP-1being 16,062 307 pg/ml, 8145 305 pg/ml, 7134 447 pg/ml, respectively. The tested substances, when given in the absence of pro-inflammatory cytokines (IL-1/TNF-), showed no regulatory effect on the expression of chemokines in HMEC-1.
In the cytokine-stimulated HMEC-1, the level of GRO- and MCP-1 in the supernatant remained similar from 0 h to 48 h, while about 20- fold stronger expression of IL-8 was detected. At 48 h, only DEX showed some down-regulatory effect on the IL-8 expression (diminished about 15 %). In HUVEC, the expression of GRO-, IL-8 and MCP-1 was significantly enhanced by 100 ng/IL-1, with increase of 17.7-, 12.5-, 3.5- folds, respectively.
Discussion
In accordance with the previous work by Goebeler et al [11], our study showed (1) the expression of IL-8 in HMEC-1was stimulated by TNF-(100 ng) and IL-1(100 ng), respectively, and TNF-(100 ng) +IL-1(100 ng) has synergistic effect, (2) the stimulation reached its maximum at 48 h, (3) the expression of GRO-, IL-8, and MCP-1 in HUVEC was also activated by TNF-(100 ng) and IL-1(100 ng), respectively. Discordant was we failed to enhance the expression of GRO- and MCP-1 in HMEC-1, even with 10 times higher levels of TNF- and IL-1. This might be due to the older passages of our cells (20-30 as compared to 3-6 in Goebeler’s work), and the different culture medium used. However, in Goebeler’s paper, most of the demonstrated data came from the experiment on primary cultures of human dermal microvascular endothelial cells (HDMEC), but not from the transformed cell line HMEC-1.
In our previous work, DEX was shown to significantly inhibit the cytokine-stimulated expression of E-selectin, VCAM-1 and ICAM-1 in HMEC-1 [9], which provided the evidence that the clinically superior anti-inflammatory effect of glucocorticoids on vasculitis was partly due to their inhibition of the CAM expression in endothelial cells. More work is needed to
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understand the regulatory effect of DEX on chemokine expression in endothelial cells, since in the present experiment only mild suppressive effect of DEX on IL-8 expression was found.The consistent finding of the basal constitutive expression of IL-8 and its enhancement in HMEC-1 under inflammation signifies its importance in the physiology of dermal microvascular endothelial cells and implies its possible role in the pathophysiology of inflammatory dermatoses [12]. The most relevant dermatoses in this context would be pyogenic granuloma (PG) and psoriasis. The histopathology of PG shows excessive capillary proliferation with large amounts of infiltrative neutrophils. Although trauma is the most usual etiology of PG, the pathogenic process is poorly defined. Based on the current data, the scenario would be (1) the traumatic event causes inflammation and stimulates the expression of vascular endothelial growth factor (VEGF) and IL-8 from keratinocytes [13, 14], (2) VEGF could modulate neutrophil transendothelial migration via up-regulation of interleukin-8 in endothelial cells to attract more neutrophils [15], (3) VEGF exerts its angiotrophic effect on the capillary formation [16]. The role of neutrophils and its relationship to vascular formation is interesting. While IL-8, MIP-2, and GRO- induced intense angiogenic reactions in vivo, and no angiogenic response to these factors was observed in neutropenic mice, demonstrating an essential role for neutrophils [17], the other experiment showed the biologically active angiostatin kringle 1-3 generated by activated human neutrophils could inhibit the basic fibroblast growth factor plus VEGF-induced endothelial cell proliferation in vitro, and both VEGF-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo [18].
In psoriasis, many observations underline the essential role of IL-8; (1) IL-8-positive neutrophils seen both in Munro's microabcesses in cases of psoriasis vulgaris and in a small spongiform pustule and much larger macropustules of Kogoj in cases of pustular psoriasis [19], (2) cultured keratinocytes of patients with psoriasis displayed much higher levels of both constitutive and induced IL-8 and IL-8 were consistently upregulated in the epidermis of patients with
psoriasis but not in lesions of patients with AD [20], (3) Response of psoriasis to interleukin-10 is associated with downregulation of the epidermal IL--8/CXCR2 pathway, (4) IL-8 is induced in skin equivalents and is highest in those derived from psoriatic fibroblasts [21]. Our result indicates in psoriatic lesions endothelial cells could serve as a possible source of IL-8, in addition to epidermis and neutrophils.
The future work is to do immunostaining of IL-8 on neutrophilic dermatoses [22], such as pyoderma gangrenosum, Sweet’s syndrome, acute generalized exanthematous pustulosis, subcorneal pustular dermatosis (Sneddon Wilkinson disease), etc. to examine if there is enhancement of IL-8 expression in dermal microvascular endothelial cells under inflammation.
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