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[Activation of mGluRI in neutrophils promotes the adherence of neutrophils to endothelial cells]

June 2010
Sheng li xue bao: [Acta physiologica Sinica] 62(3):219-24

June 2010
62(3):219-24

Source
PubMed

Authors:

Jinfeng Li

Zhengzhou University

Show all 11 authorsHide

L-glutamate (Glu) is an excitatory neurotransmitter in the mammalian central nervous system. Relatively much attention has been paid to functional expression of Glu signaling molecules in peripheral tissues very recently. The present study tested the hypothesis that the activation of group I metabotropic glutamate receptor (mGluRI) in neutrophils stimulated neutrophils adherence to endothelial cells by increasing the surface expression of certain adhesion molecules. Peripheral blood was obtained by venipuncture from healthy donors, and the neutrophils were isolated by Ficoll-Hypaque gradient centrifugation. Neutrophils floating into DMEM/F12 culture medium containing 10% fetal bovine serum were then used immediately. Immunocytochemistry and real-time quantitative RT-PCR were used to detect the expression of mGluRI (mGluR1 and mGluR5) in neutrophils. The adherence of neutrophils to cultured human normal umbilical vein endothelial cells (HUVE-12) was measured by the colorimetric method. Cell surface expression of adhesion molecule CD11a in the neutrophils was determined by flow cytometry. Immunocytochemistry and real-time quantitative RT-PCR showed that mGluR1 and mGluR5 were constitutively expressed in neutrophils. Application of mGluRI agonist S-3,5-dihydroxyphenylglycine (S-DHPG) (1x10(-8)-1x10(-6) mol/L) showed a dose-dependent stimulatory effect on the adherence of neutrophils to HUVE-12 (P<0.05 or P<0.01), with a maximum effect at 1x10(-6) mol/L (P<0.01). Incubations as short as 30 min were sufficient to induce increased adherence after the beginning of S-DHPG treatment. Following time extension (0.5-5 h), S-DHPG (1x10(-6) mol/L) increased the rate of neutrophils adhesion to HUVE-12 with a maximum effect at 0.5 h (P<0.01). However, a time-dependent effect of S-DHPG on the rate of neutrophils adhesion to HUVE-12 was not observed during the experimental period. 1x10(-6) mol/L of S-DHPG also induced an increased surface expression of adhesion molecule CD11a (P<0.01) when neutrophils were preincubated with 1x10(-6) mol/L of S-DHPG for 1 h. Furthermore, the specific mGluRI antagonist (RS)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG, 0.5 mmol/L) significantly abolished the stimulatory effect of S-DHPG (1x10(-6) mol/L) on the adherence of neutrophils to HUVE-12 (P<0.01). These results suggest that the activation of mGluRI in neutrophils results in increased adhesion molecule CD11a expression and thereby promotes the adherence of neutrophils to endothelial cells.

. mGluR1 and mGluR5 mRNA expression on neutrophils

…

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生理学报 Acta Physiologica Sinica, June 25, 2010, 62 (3): 219-224

http://www.actaps.com.cn 219

研究论文

中性粒细胞 mGluRI 的激活促进其与内皮细胞相互黏附

刘雪云

1,2

，刘永

，李金凤

，岳少杰

，申丽

，李晨

，汉建忠

，许建平

，冯丹丹

，刘惠君

，

罗自强

1,*

中南大学湘雅医学院生理学系，长沙 410078；

福建中医药大学中西医结合系生理教研室，福州 350108；

中南大学

湘雅医院儿科，长沙 410078

摘要：本文旨在探讨 I组代谢型谷氨酸受体(group I metabotropic glutamate receptor, mGluRI)在中性粒细胞上的表达，以及

该受体的激活对中性粒细胞与内皮细胞相互黏附的影响。取健康人新鲜静脉血，Ficoll-Hypaque 密度梯度离心法分离中性

粒细胞，免疫细胞化学法和 real-time PCR 法检测中性粒细胞 mGluRI (包括 mGluR1 和mGluR5)的表达，分别应用不同浓度

的mGluRI特异性激动剂 S-3, 5- 二羟基苯甘氨酸(S-3,5-dihydroxy-phenylglycine, S-DHPG)处理中性粒细胞不同时间，通过比

色法检测中性粒细胞和人正常脐静脉内皮细胞(human normal umbilical vein endothelial cells, HUVE-12)黏附率，采用流式细胞

术测定中性粒细胞黏附分子 CD11a表达的变化。结果证实中性粒细胞表达 mGluRI (mGluR1/5)；S-DHPG在1×10

-8

~1×10

-6

mol/L

浓度范围内呈剂量依赖性地提高中性粒细胞与内皮细胞之间的黏附率(P<0.05 或P<0.01)；1×10

-6

mol/L S-DHPG单独作用于

中性粒细胞 0.5 h，即可促进中性粒细胞黏附于内皮细胞(P<0.01) ，但没有时间依赖性；1×10

-6

mol/L S-DHP G 单独作用于

中性粒细胞可促进 CD11a 表达(P<0.01)；mGluRI拮抗剂(RS)-

α-

甲基 -4- 羧基苯甘氨酸[(RS)-

-methyl-4-carboxyphenylglycine,

(±)-MCPG] (0.5 mmol/L)可以显著阻断激动剂 S-DHPG (1×10

-6

mol/L, 1 h)的促黏附效应(P<0.01)。上述结果证实了中性粒细胞

膜上 mGluRI的激活可增强中性粒细胞表面黏附分子 CD11a的表达，促进中性粒细胞与内皮细胞的黏附。

关键词：代谢型谷氨酸受体 1；代谢型谷氨酸受体5；中性粒细胞；血管内皮细胞；细胞黏附分子；CD11a

中图分类号：R331.1+42

Activation of mGluRI in neutrophils promotes the adherence of neutrophils to

endothelial cells

LIU Xue-Yun

1,2

, LIU Yong

, LI Jin-Feng

, YUE Shao-Jie

, SHEN Li

, LI Chen

, HAN Jian-Zhong

, XU Jian-Ping

FENG Dan-Dan

, LIU Hui-Jun

, LUO Zi-Qiang

1,*

Department of Physiology, Xiangya School of Medicine, Central South University, Changsha 410078, China;

Fujian University of

Traditional Chinese Medicine, Fuzhou 350108, China;

Department of Pediatrics, Xiangya Hospital, Central South University,

Changsha 410078, China

Abstract: L-glutamate (Glu) is an excitatory neurotransmitter in the mammalian central nervous system. Relatively much attention has

been paid to functional expression of Glu signaling molecules in peripheral tissues very recently. The present study tested the

hypothesis that the activation of group I metabotropic glutamate receptor (mGluRI) in neutrophils stimulated neutrophils adherence

to endothelial cells by increasing the surface expression of certain adhesion molecules. Peripheral blood was obtained by venipuncture

from healthy donors, and the neutrophils were isolated by Ficoll-Hypaque gradient centrifugation. Neutrophils floating into DMEM/

F12 culture medium containing 10% fetal bovine serum were then used immediately. Immunocytochemistry and real-time quantitative

RT-PCR were used to detect the expression of mGluRI (mGluR1 and mGluR5) in neutrophils. The adherence of neutrophils to cultured

human normal umbilical vein endothelial cells (HUVE-12) was measured by the colorimetric method. Cell surface expression of

adhesion molecule CD11a in the neutrophils was determined by flow cytometry. Immunocytochemistry and real-time quantitative RT-

PCR showed that mGluR1 and mGluR5 were constitutively expressed in neutrophils. Application of mGluRI agonist S-3,5-

Received 2010-01-18 Accepted 2010-03-15

The work was supported by the National Natural Science Foundation of China (No. 30370531, 30471835).

Corresponding author. Tel: +86-731-82355051; Fax: +86-731-82355056; E-mail: luozq1962@163.com

生理学报 Acta Physiologica Sinica, June 25, 2010, 62(3): 219-224

220

dihydroxyphenylglycine (S-DHPG) (1×10

-8

-1×10

-6

mol/L) showed a dose-dependent stimulatory effect on the adherence of neutro-

phils to HUVE-12 (P<0.05 or P<0.01), with a maximum effect at 1×10

-6

mol/L (P<0.01). Incubations as short as 30 min were sufficient

to induce increased adherence after the beginning of S-DHPG treatment. Following time extension (0.5-5 h), S-DHPG (1×10

-6

mol/L)

increased the rate of neutrophils adhesion to HUVE-12 with a maximum effect at 0.5 h (P<0.01). However, a time-dependent effect of

S-DHPG on the rate of neutrophils adhesion to HUVE-12 was not observed during the experimental period. 1×10

-6

mol/L of S-DHPG

also induced an increased surface expression of adhesion molecule CD11a (P<0.01) when neutrophils were preincubated with 1×10

-6

mol/L of S-DHPG for 1 h. Furthermore, the specific mGluRI antagonist (RS)-α-methyl-4-carboxyphenylglycine ((±)-MCPG, 0.5

mmol/L) significantly abolished the stimulatory effect of S-DHPG (1×10

-6

mol/L) on the adherence of neutrophils to HUVE-12 (P<0.01).

These results suggest that the activation of mGluRI in neutrophils results in increased adhesion molecule CD11a expression and

thereby promotes the adherence of neutrophils to endothelial cells.

Key words: metabotropic glutamate receptor 1; metabotropic glutamate receptor 5; neutrophils; vascular endothelial cells; cell adhesion

molecules; antigens, CD11a

谷氨酸(glutamate, Glu)是脊椎动物中枢神经系统

中一类重要的兴奋性神经递质，通过激活谷氨酸受

体(glutamate receptors, GluRs)发挥作用。Glu过度

激活其受体所引起的兴奋性神经毒性在神经损伤的

发生中具有重要作用

[1-3]

。GluRs可分为亲离子型谷

氨酸受体(inotropic GluRs, iGluRs)和亲代谢型谷氨酸

受体(metabotropic GluRs, mGluRs)两类。I组代谢

型谷氨酸受体(group I metabotropic glutamate receptor,

mGluRI)是mGluRs 的重要亚型。近年来一些研究者

注意到在中枢神经系统外的外周非神经组织也广泛

存在 GluRs的表达

[4-7]

，但它们的生理及病理意义远

未阐明。

急性肺损伤(acute lung injury, ALI)和急性呼吸窘

迫综合征(acute respiratory distress syndrome, ARDS)

是常见的肺部严重疾病，发病机制尚未完全阐明。

肺内中性粒细胞的滞留、激活以及与血管内皮细胞

的黏附在 ALI和ARDS 的发生发展中起关键性的作

用

[8]

。ALI 和ARDS 是一种失控的炎症反应，以肺

毛细血管内皮细胞及肺泡上皮细胞广泛损伤为病理

特点。中性粒细胞与内皮细胞的黏附是炎症反应的

最初现象。本研究室的前期研究显示，腹腔注射

Glu可导致 ALI，且肺内有大量中性粒细胞的渗出

[9]

，

进一步显示iGluRs中的N-甲基-D-天冬氨酸(NMDA)

受体的激活在此过程中起着重要作用

[10]

。在中性粒

细胞浸润过程中，中性粒细胞与血管内皮细胞之间

的黏附是中性粒细胞浸润中非常重要的第一步，但

mGluRs 在此过程中有何影响未见报道。本实验通

过观察 mGluRI 在中性粒细胞的表达，以及该受体

的激活对中性粒细胞与内皮细胞黏附的影响，对其

可能的机制进行探讨，为深入研究肺脏等外周组织

中GluRs的生理及病理意义，拓展肺部疾病机制及

防治的研究提供新的思路。

1 材料和方法

1.1 材料　　人正常脐静脉内皮细胞株(HUVE-12,

ATCC细胞库, CRL-2480

)由中南大学湘雅医学院细

胞室提供，人新鲜全血来自健康志愿者。DMEM/

F12 培养基(GIBCO, USA)；人中性粒细胞和淋巴细

胞分离液(灏洋生物制品有限公司，天津)；4- 硝基

酚-N-乙酰 -β-D-氨基葡萄糖苷(4-nitrophenol-N-

acetyl-β-D-glucose-aminide, PNP-NAG, Sigma,

USA)；mGluRI 特异性激动剂 S-3,5- 二羟基苯甘氨

酸(S-3,5-dihydroxy-phenylglycine, S-DHPG, Sigma,

USA)；mGluRI 的特异性拮抗剂(RS)-α-甲基 -4- 羧基

苯甘氨酸[(RS)-α-methyl-4-carboxyphenylglycine, (±)-

MCPG, Sigma, USA]；DEPC (Sigma, USA)；即用

型SP 免疫组化试剂盒、DAB染色试剂盒(中杉金桥

生物技术有限公司，北京)；胎牛血清(fetal bovine

serum，FBS，四季青生物工程材料有限公司，杭

州)；兔抗人 mGluR1 抗体和兔抗人 mGluR5 抗体

(Abcam, England)；PE 标记小鼠抗人 CD11a 单克隆

抗体(BioLegend, USA)；引物探针，RT 试剂，荧光

定量 PCR 试剂(闪晶分子生物科技有限公司，上海)。

1.2 Ficoll-Hypaque 密度梯度离心法分离人中性粒

细胞与实验分组　　参考文献方法

[11]

取新鲜抗凝血

与PBS 液体 1:1 混合后，加于等体积的人中性粒细

胞分离液界面上，2 500 r/min 水平离心 20 min后，

取第 2、3层，用 PBS 洗涤，FBS 重悬沉淀加于等

体积人淋巴细胞分离液界面上，2 500 r/min水平离

心20 min后，取第 3层，用 PBS 洗涤后台盼蓝染

色计数活细胞>99%，瑞吉染色计数纯度达到 90%

以上。细胞沉淀悬浮于含 10% FBS的DMEM/F12

221

刘雪云等：中性粒细胞 mGluRI的激活促进其与内皮细胞相互黏附

培养基中，新鲜应用。

新鲜分离的中性粒细胞分为对照组和 mGluRI特

异性激动剂 S-DHPG 组。为观察 S-DHPG 预处理中

性粒细胞对黏附变化率影响的量效和时效关系，实

验组加入终浓度为 1×10

-8

、1×10

-7

、1×10

-6

mol/L 的

S-DHPG 分别孵育 1 h，测定黏附率；另加入 1×10

-6

mol/L S-DHPG分别孵育 0.5~5 h，测定黏附率。部

分实验分为对照组、(±)-MCPG (0.5 mmol/L)组、S-

DHPG (1×10

-6

mol/L)和(±)-MCPG (0.5 mmol/L) + S-

DHPG (1×10

-6

mol/L)组；作用 1 h，测定黏附率。

对照组不加药，只加等体积量培养基。

1.3 HUVE-12细胞的培养

[12]

及与中性粒细胞黏附

率的测定

[13]

　　HUVE-12的培养采用含10% FBS的

DMEM/F12 培养液。将内皮细胞 1×10

个/L 接种于

96 孔板中，每孔 100 μL，次日换无血清培养基，

第三天实验前再换新鲜无血清培养基。分离中性粒

细胞稀释为 1×10

个/L，将预处理的中性粒细胞以

每孔 100 μL加入内皮细胞培养孔中继续培养 2 h，

PBS 洗涤 3次，去除未黏附的中性粒细胞，每孔加

入7.5 mmol/L PNP-NAG (溶于 0.1 mol/L柠檬酸缓冲

液，pH 5.0) 60 μL，置 37 ºC、5% CO

培养箱中。2 h

后每孔加入 90 μL甘氨酸缓冲液(甘氨酸 50 mmol/L、

EDTA 5 mmol/L，pH 10.4)终止反应，酶标仪 405

nm测吸光度(A)值。计算黏附率，黏附率 =(黏附细

胞A值−HUVE-12细胞 A值)/加入总细胞A值×100%。

采用黏附变化率作为测定指标，黏附变化率 =加药

组黏附率 /对照组黏附率 ×100%。

1.4 免疫细胞化学检测中性粒细胞的mGluRI (mGluR1

和mGluR5)蛋白表达　　将提取的中性粒细胞涂

片，冰浴纯甲醇固定。参考 SP 免疫组化试剂盒说

明书，3% H

消除内源性过氧化物酶活性，相继

滴加 1:10 稀释兔抗人 mGluR1 (或mGluR5)一抗及山

羊抗兔二抗 37 ºC 孵育，DAB 法显色后镜下观察。

1.5 Real-time PCR测定中性粒细胞 mGluR1 及mGluR5

的mRNA 表达　　按2×10

个中性粒细胞中加 1 mL

Trizol 提取总 RNA，由上海闪晶分子生物科技有限

公司完成中性粒细胞 mGluR1 及mGluR5 表达水平的

荧光定量 PCR 测定。基因引物序列如下：mGluR1

上游：5’-GTCGGGCTCCTTTTGTTTTT-3’，下

游：5’-GAAGAGGGCTCCAATGATGAC-3’，

mGluR1 probe：FAM+TTCCCAGCGATCTTTTT-

GGAGGTG+TAMRA；mGluR5 上游：5’-AATCT-

CCCGATGTCAAGTGGT-3’，下游：5’-TGTTGTA-

TTTGCTGTTCTCCTGTG-3’，mGluR5 probe：

FAM+CCAGAAACAAACCACCGAAACCCTT+

TAMRA ；GAPDH 上游：5’-CCACTCCTCCACC-

TTTGAC-3’，GAPDH 下游：5’-ACCCTGTTGC-

TGTAGCCA-3’，GAPDH-PROBE：FAM+TTG-

CCCTCAACGACCACTTTGTC+TAMRA。RT 反应

体系(20 μL)：2×First-Strand Buffer 10 μL，随机引

物(100 pmol/μL) 1 μL，RT-mix 1 μL，RNA 8

μL；条件：25 ºC 10 min，40 ºC 50 min，90 ºC

5 min。荧光定量 PCR 反应体系(50 μL)：2×Hotstart

Fluo-PCR mix 25 μL，Primers (25 pmol/μL) 0.8

μL×2，Probe (25 pmol/μL) 0.3 μL， cDNA 1 μL，

DEPC 水22.1 μL；扩增条件：94 ºC 4 min，94 ºC

20 s，60 ºC 30 s，循环 40 次。

1.6 流式细胞术测定中性粒细胞表面黏附分子CD11a

(LFA-1)的表达　　实验分为空白对照组和S-DHPG

(1×10

-6

mol/L)组。参照文献方法

[14]

及一抗说明书，

取新鲜全血 120 μL分别加各处理因素孵育 1 h。为

了避免体外激活，直接在全血中加 CD11a (PE 标

记)单克隆抗体避光孵育 30 min，然后加 1 mL等

渗红细胞裂解液，离心后用含 1% 牛血清白蛋白的

PBS 洗涤后重悬细胞，流式细胞仪根据前向散射和

侧向散射光线特征向中性粒细胞开门，测阳性细胞

百分率及细胞的平均荧光强度(median fluorescence

intensity, MFI)。

1.7 统计学分析　　所有数据用 SPSS13.0 统计软

件进行统计，测定结果以 mean±SD 表示。两组间

比较用成组 t检验；多组比较采用单因素方差分析

进行显著性检验，方差齐同，两两比较采用 LSD 检

验；方差不齐，两两比较则采用 Dunnett’s T3检

验。P<0.05 为差异有统计学意义。

2 结果

2.1 中性粒细胞 mGluRI (mGluR1 和mGluR5)的

表达

免疫细胞化学检测显示中性粒细胞均可见

mGluR1及mGluR5的蛋白表达(图1)。Real-time PCR

也证实，中性粒细胞存在 mGluR1 及mGluR5 的

mRNA 表达(表1)。

2.2 S-DHPG 对中性粒细胞与内皮细胞相互黏附

的影响

2.2.1 不同剂量 S-DHPG 对中性粒细胞与内皮细

胞相互黏附的影响

生理学报 Acta Physiologica Sinica, June 25, 2010, 62(3): 219-224

222

为探讨mGluRI的激活对中性粒细胞与内皮细胞

相互黏附的影响，实验预先给予中性粒细胞不同浓

度mGluRI 的激动剂S-DHPG (1×10

-8

~1×10

-6

mol/L)孵

育1 h，测定与 HUVE-12细胞的黏附率的变化。发

现当 S-DHPG单独作用于中性粒细胞时，在 1×10

-8

1×10

-6

mol/L 浓度范围内，随着 S-DHPG浓度增高，中

性粒细胞黏附于内皮细胞的黏附率增高(P<0.05 或P<

0.01)，其中以 1×10

-6

mol/L 的促黏附效果最强(图2)。

2.2.2 S-DHPG 不同作用时间对中性粒细胞与内皮

细胞相互黏附的影响

给予中性粒细胞 1×10

-6

mol/L S-DHPG 后孵育

0.5~5 h，发现当 S-DHPG单独作用于中性粒细胞仅

0.5 h，即可促进中性粒细胞黏附于内皮细胞(P<

0.01)，随着作用时间的延长，中性粒细胞黏附于

内皮细胞的比率并不随之增加(图3)。

2.3 mGluRI受体阻断剂对中性粒细胞与内皮细

胞相互黏附的影响

为进一步确认mGluRI受体激活对中性粒细胞与

内皮细胞相互黏附的影响，本研究观察了 mGluRI

的特异性拮抗剂(±)-MCPG对激动剂S-DHPG促黏附

作用的影响，以排除 S-DHPG可能的其它非受体激

动机制。结果显示，单纯(±)-MCPG (0.5 mmol/L)

对黏附率无明显影响，但可有效抑制激动剂 S-DHPG

促黏附作用(P<0.01) (图4)。

2.4 mGluRI 的激活对中性粒细胞表面黏附分子

CD11a (LFA-1)表达的影响

流式细胞术检测显示，mGluRI 激动剂 S-DHPG

(1×10

-6

mol/L)预处理中性粒细胞 1 h，CD11a (LFA-1)

的阳性率和荧光强度与对照组比较均显著增加

图 1. 中性粒细胞 mGluR1 与mGluR5 的蛋白表达

Fig. 1. The protein expression of mGluR1 and mGluR5 on neutrophils stained by SP method. A: The expression of GluR1 on neutrophils.

B: The expression of GluR5 on neutrophils. Scale bar, 10 μm.

表1. 中性粒细胞 mGlu R1 及mGluR5 mRNA 的表达(real-time

PCR)

Table 1. mGluR1 and mGluR5 mRNA expression on neutrophils

detected by real-time PCR

Ct Copy number Gene expression ratio (%)

GAPDH 22.24 3.86×10

GluR1 27.44 7.16×10

GluR5 24.69 5.88×10

GluR1/GAPDH 1.855

GluR5/GAPDH 15.233

图 2. 不同浓度 S-DHPG (1×10

-8

~1×10

-6

mol/L)对中性粒细胞

与内皮细胞相互黏附的影响

Fig. 2. Effects of different concentrations of S-DHPG (1×10

-8

1×10

-6

mol/L) on the adhesion of neutrophils to HUVE-12. Neu-

trophils were pretreated with S-DHPG (1×10

-8

-1×10

-6

mol/L) for

1 h. mean±SD, n=5.

P<0.05,

P<0.01 compared with control;

0.05,

P<0.01 compared with 1×10

-8

mol/L group.

223

刘雪云等：中性粒细胞 mGluRI的激活促进其与内皮细胞相互黏附

(P<0.01)。说明 mGluRI 的激活可以明显提高中性

粒细胞表面 CD11a 的表达(表2)。

3 讨论

Glu的mGluRs 属于 G蛋白耦联受体，通过调

节细胞内第二信使的产生而影响细胞的功能活动。

mGluRs 家族根据结构同源性、药理学特性和传导

通路可分为三组 8个亚型，I组mGluRs 包括mGluR1

和mGluR5；II 组包括 mGluR2 和mGluR3；III组

包括 mGluR4、mGluR6、mGluR7 和mGluR8

[15,16]

。

I组mGluRs 过度激活在神经损伤的发生中有着重要

地位

[17]

。在外周，如在心脏、血液中的淋巴细胞、

人脑及皮肤微血管内皮上、胰腺和消化道等组织器

官中也发现有 mGluRI 表达

[5-7,18-20]

，提示 Glu可通过

mGluRI 对这些器官发挥某些调节作用。在脑微血

管内皮上的 mGluRI 被Glu 激活后，引起血管通透

性的升高，提示 mGluRI 的激活可能参与疾病过程

中微血管通透性增高的发生过程

[7]

。mGluRI 在中性

粒细胞上是否存在未见报道，本研究通过采用免疫

细胞化学和 real-time PCR的方法在蛋白和 mRNA 水

平证实，中性粒细胞存在 mGluRI 的mGluR1 和

mGluR5 两个亚型的表达。

为揭示 mGluRI 的激活对中性粒细胞功能的调

节，本实验采用 mGluRI 选择性激动剂 S-DHPG和

选择性拮抗剂(±)-MCPG 探讨中性粒细胞上 mGluRI

的激活对中性粒细胞与 HUVE-12相互黏附的影响。

结果显示，S-DHPG在1×10

-8

~1×10

-6

mol/L 范围内呈

浓度依赖方式促进中性粒细胞与内皮细胞的黏附，

该效应可被 mGluRI 的选择性拮抗剂(±)-MCPG 所阻

断。提示中性粒细胞上的 mGluRI 激活可促进中性

粒细胞与内皮细胞的相互黏附。

中性粒细胞必须首先和微血管内皮细胞黏附才

能跨血管迁移并在炎症部位聚集，因此在二者的黏

附中，产生的黏附分子及作用是极为关键的一

步。在中性粒细胞与内皮细胞黏附的早期阶段中，

选择素家族与整合素家族黏附分子发挥重要作用。

当炎症介质使内皮细胞和中性粒细胞被进一步激活

时，内皮细胞表面的细胞间黏附分子 l (intercellular

adhesion molecule 1, ICAM-1)、中性粒细胞表面的

整合素 α

(淋巴细胞功能相关抗原 -1，lymphocyte-

function-associated antigen-1, LFA-1, 又称 CD11a)的

表达增强或活化

[21]

。中性粒细胞表面的 LFA-1 可与

血管内皮细胞上的 ICAM-1 结合，使中性粒细胞停

图 3. S-DHPG (1×10

-6

mol/L)不同作用时间对中性粒细胞与内

皮细胞相互黏附的影响

Fig. 3. Effects of S-DHPG (1×10

-6

mol/L) treatment for different

time on the adhesion of neutrophils to HUVE-12. mean±SD, n=5.

P<0.01 vs Control;

P<0.05,

P<0.01 vs 0.5 h group;

P<0.05

vs 3 h group.

图 4. (±)-MCPG (0.5 mmol/L)对S-DHPG 促中性粒细胞与

HUVE-12 细胞黏附率的影响

Fig. 4. Effects of (±)-MCPG (0.5 mmol/L) on adhesion rate of

neutrophils and HUVE-12 promoted by S-DHPG. (±)-MCPG

group: (±)-MCPG (0.5 mmol/L) pretreatment for 1 h; S-DHPG

group: S-DHPG (1×10

-6

mol/L) pretreatment for 1 h; (±)-MCPG +

S-DHPG group: pretreatment of both (±)-MCPG (0.5 mmol/L)

and S-DHPG (1×10

-6

mol/L) for 1 h. mean±SD, n=5.

P<0.01 vs

Control;

P<0.01 vs (±)-MCPG;

P<0.01 vs S-DHPG.

表2. mGluRI的激活对中性粒细胞黏附分子 CD11a (LFA-1)表

达的影响

Table 2 . Effects of the mGluRI activation on the expression of CD11a

(LFA-1) in neutrophils detected by flow cytometry

Group Positive rate (%) MFI

Control 40.15±3.03 27.20±2.86

S-DHPG 56.45±4.95

35.58±2.13

MFI: median fluorescence intensity. mean±SD, n=3.

P<0.01 vs

control group.

生理学报 Acta Physiologica Sinica, June 25, 2010, 62(3): 219-224

224

止滚动而黏附在内皮细胞上，进而通过白细胞的变

形，伸出伪足并插入内皮细胞的间隙，然后以阿米

巴运动的形式到达周围炎症组织

[22]

。本实验采用流

式细胞术测定中性粒细胞表面的 CD11a 表达变化。

当用 1×10

-6

mol/L S-DHPG处理全血 1 h后CD11a 阳

性细胞率明显升高，MFI增加，说明 mGluRI 的激

活可增强中性粒细胞表面的 CD11a表达，这可能是

mGluRI的激活促进中性粒细胞与内皮细胞相互黏附

的分子机制。这一结果提示 Glu可通过 mGluRI 的

激活参与外周炎症过程中性粒细胞与内皮细胞相互

黏附的调控，进一步扩大了 Glu 外周非神经组织损

伤机制的认识，也为临床 ALI、ARDS 等肺部疾病

防治的研究提供了新的思路。

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Nov 2000
NEUROSCI LETT

Stimulation of group I metabotropic glutamate receptors (mGluR 1 and 5) activates G-protein coupled-phospholipase C (PLC) to release 1,2-diacylglycerol (DAG) and arachidonic acid (ArAc). To elucidate the role of group I mGluR, we tested the effects of (S)-alpha-methyl-4-carboxy-phenylglycine (MCPG, mGluR 1 and 5 antagonist), 1-aminoindan-1,5-dicarboxylic acid (AIDA, mGluR 1a specific antagonist) and 2-methyl-6-(phenylethynyl) pyridine (MPEP, mGluR 5 antagonist) on ArAc release and neuronal survival after transient forebrain ischemia in gerbils. Ischemia resulted in (a) significant release of ArAc at 1-day reperfusion and (b) significant neuronal death in the hippocampal CA1 subfield after 6-day reperfusion. MCPG and MPEP decreased ArAc release and also significantly increased neuronal survival. AIDA was less effective in decreasing ArAc release and had no effect on neuronal death. These results suggest that activation of mGluR 5 may be an important pathway in ArAc release and neuronal death after transient ischemia.

Group I metabotropic glutamate receptors prevent endothelial programmed cell death independent from MAP kinase p38 activation in rat

Article

Mar 2001
NEUROSCI LETT

Activation of Group I metabotropic glutamate receptors (mGluRs) prevents neuronal programmed cell death (PCD), but the role of these receptors in the vascular endothelial cell (EC) system has not been defined. Since ECs are principal targets for ischemic free radical injury, we examined whether the mGluR system could modulate vascular PCD. Activation of the Group I mGluR system, but not antagonism, addressed two distinct pathways of PCD by preventing the destruction of genomic DNA and maintaining EC membrane asymmetry. The induction of nitric oxide (NO)-induced PCD in ECs paralleled the specific activation of the MAP kinase p38 pathway, but the vascular protection conferred by the Group I mGluR system appears to rely on more downstream cellular pathways. We provide initial evidence for Group I mGluRs to prevent NO-induced vascular injury and offer new directions for vascular disease treatment.

Glutamate signaling in non-neuronal tissues

Article

May 2001
TRENDS PHARMACOL SCI

Since the discovery of its role in the CNS, glutamate, together with its involvement in signalling at synapses, has been the subject of a vast amount of research. More recently, it has become clear that glutamate signalling is also functional in non-neuronal tissues and occurs in sites as diverse as bone, pancreas and skin. These findings raise the possibility that glutamate acts as a more widespread 'cytokine' and is able to influence cellular activity in a range of tissue types. The impact of these discoveries is significant because they offer a rapid way to advance the development of therapeutics. Agents developed for use in neuroscience applications might be beneficial in the modulation of pathology peripherally, impacting on conditions such as osteoporosis, diabetes and wound healing.

Inhibiton of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune disease

Article

Mar 2002

This review focuses on providing insights into the structural basis and clinical relevance of LFA-1 and VLA-4 inhibition by peptides and small molecules as adhesion-based therapeutic strategies for inflammation and autoimmune diseases. Interactions of cell adhesion molecules (CAM) play central roles in mediating immune and inflammatory responses. Leukocyte function-associated antigen (LFA-1, alpha(L)beta(2), and CD11a/CD18) and very late antigen (VLA-4, alpha(4)beta(1), and CD49d/CD29) are members of integrin-type CAM that are predominantly involved in leukocyte trafficking and extravasation. LFA-1 is exclusively expressed on leukocytes and interacts with its ligands ICAM-1, -2, and -3 to promote a variety of homotypic and heterotypic cell adhesion events required for normal and pathologic functions of the immune systems. VLA-4 is expressed mainly on lymphocyte, monocytes, and eosinophils, but is not found on neutrophils. VLA-4 interacts with its ligands VCAM-1 and fibronectin (FN) CS1 during chronic inflammatory diseases, such as rheumatoid arthritis, asthma, psoriasis, transplant-rejection, and allergy. Blockade of LFA-1 and VLA-4 interactions with their ligands is a potential target for immunosuppression. LFA-1 and VLA-4 antagonists (antibodies, peptides, and small molecules) are being developed for controlling inflammation and autoimmune diseases. The therapeutic intervention of mostly mAb-based has been extensively studied. However, due to the challenging relative efficacy/safety ratio of mAb-based therapy application, especially in terms of systemic administration and immunogenic potential, strategic alternatives in the forms of peptide, peptide mimetic inhibitors, and small molecule non-peptide antagonists are being sought. Linear and cyclic peptides derived from the sequences of LFA-1, ICAM-1, ICAM-2, VCAM-1, and FN C1 have been shown to have inhibitory effects in vitro and in vivo. Finally, understanding the mechanism of LFA-1 and VLA-4 binding to their ligands has become a fundamental basis in developing therapeutic agents for inflammation and autoimmune diseases.

[Activation of mGluRI in neutrophils promotes the adherence of neutrophils to endothelial cells]

Abstract and Figures

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