Acta Physiologica Sinica, April 25, 2003, 55(2): 121-127

Received 2002-07-08

Accepted 2002-10-09

This project was supported by the National Natural Science Foundation of China (No. 30270586, 39800053).

Corresponding author. Tel: +86-731-2650401; E-mail: ttyyrr3668@sohu.com

Research Paper

Regulatory peptides modulate ICAM-1 gene expression and NF-κB activity in bronchial epithelial cells

TAN Yu-Rong, QIN Xiao-Qun*, GUAN Cha-Xiang, ZHANG Chang-Qing, LUO Zi-Qiang,

SUN Xiu-HongDepartment of Physiology, Xiangya School of Medicine, Central South University, Changsha 410078

Abstract: Intercellular adhesion molecule-1 (ICAM-1) is an important adhesion molecule leading to adhesion between cells; NF-κB, being universally distributed in the organism, is an important nuclear transcription factor leading to a rapid response to the stimuli. Line of evidence have shown that ICAM-1 transcription and NF-κB activation is an important step of inflammatory reaction. To testify that intrapulmonary regulatory peptides modulate inflammatory lesion of bronchial epithelial cells (BECs) through their effect on ICAM-1 expression and nuclear factor κB (NF-κB) activation, we used immunocytochemistry, RT-PCR, and electrophoretic mobility-shift assay (EMSA) to determine the ICAM-1 expression and NF-κB activity in BECs. The effects of NF-κB inhibitor MG-132 on ICAM-1 expression were also observed. The results showed that vasoactive intestinal peptide (VIP) and epidermal growth factor (EGF) decreased ICAM-1 expression in O3-stressed BECs, while endothelin-1 (ET-1) and calcitonin gene-related peptides (CGRP) increased ICAM-1 expression in resting BECs. MG-132 blocked ICAM-1 expression induced by O3, ET-1 and CGRP. The results obtained by using EMSA confirmed that VIP and EGF restrained the activation of NF-κB in O3-stressed BECs; CGRP and ET-1 promoted activation of NF-κB. These observations indicate that VIP and EGF abated the injury by means of down-regulatory effects on ICAM-1 transcription and NF-κB activation, while ET-1 and CGRP enhanced the inflammation reaction by an up-regulatory effect. It is suggested that a developing and intensive airway inflammation correlates closely with a persistent expression of ICAM-1 and repeated activation of NF-κB.

Key words: intrapulmonary regulatory peptides; intercellular adhesion molecule-1 (ICAM-1); nuclear factor κB (NF-κB); bronchial epithelial cells

肺内调节肽对支气管上皮细胞ICAM-1表达及NF-κB活性的调控

谭宇蓉, 秦晓群*, 管茶香, 张长青, 罗自强, 孙秀泓

中南大学湘雅医学院生理学教研室, 长沙 410078

摘要: 细胞间粘附分子-1 (ICAM-1)是介导细胞与细胞之间粘附的重要生物分子; 核因子-κB (NF-κB)是体内普遍存在、能迅速对刺激产生反应的重要核转录因子。越来越多的证据显示, ICAM-1表达与NF-κB激活是炎症反应的重要步骤。我们应用免疫组化、RT-PCR、凝胶阻滞电泳(EMSA)等多种实验方法, 观察了肺内调节肽对支气管上皮细胞ICAM-1表达及NF-κB活性的影响, 以及NF-κB抑制剂MG-132对ICAM-1表达的影响。实验结果发现, VIP、EGF可使臭氧应激BECs的ICAM-1表达降低; ET-1、CGRP可使未受应激BECs的ICAM-1表达增加。NF-κB抑制剂MG-132可阻断O3、ET-1、CGRP引起的ICAM-1表达, 提示NF-κB在调控ICAM-1表达中起重要作用。 EMSA结果显示, BECs中NF-κB在臭氧应激下反复激活,CGRP与ET-1可促进NF-κB的激活; VIP与EGF可抑制臭氧应激的BECs中NF-κB的激活。以上结果说明, VIP、EGF可通过下调ICAM-1转录及NF-κB激活减轻炎症反应, 而ET-1、CGRP可通过上调ICAM-1转录及NF-κB激活、加大炎症反应。ICAM-1与NF-κB的持续表达和反复激活是炎症持续加重发展的重要因素。

关键词: 肺内调节肽; 细胞间粘附分子-1; 核因子-κB; 支气管上皮细胞

中图分类号: Q471; R332.2

Bronchial epithelium is an important barrier against the environmental injury factors. Injury or function defect of bronchial epithelial cells (BECs) may be the initial step for the development of airway hyperresponsiveness. BEC is also a kind of important proinflammatory cells. When BECs are stressed or stimulated, they can release interleukins or other inflammatory media, express adhesion molecules, adhere to and activate inflammation-responding cells, and then transmit inflammation injury signals to leukocytes. Adhering to BECs and being activated are the basic factors for the activation of polymorphonuclear leukocytes (PMNs) and eosinophils (EOS) for this inflammation signal transmission. Previous experiments in our lab showed that the adhesion of PMNs or EOS to BECs was mediated by intercellular adhesion molecule-1 (ICAM-1)[1]. The interaction between ICAM-1 and its ligands mediates a firm adhesion between leukocytes and BECs, which enables leukocytes to be recruited and anchored on the inflamed tissue. However, the regulatory factors that may take part in ICAM-1 expression during airway diseases and the involved signal transduction pathway are still not clear.

Nuclear factor κB (NF-κB), an important transcriptional activator, plays a crucial role in the host′s response to infection. Evidence shows the activation of NF-κB is the important step in inflammation reaction. Many nuclear transcription factors such as Sp-1, NF-1, AP-1 and NF-κB etc., are closely related to transcriptional activation of various inflammation medium coding genes. The activation of NF-κB correlates with the phosphorylation of inhibitoryκB (IκB) and subsequent degradation of IκB. Activated NF-κB then binds to the cognate DNA binding sites and activates transcription of target gene. The present study was carried out to testify which transcriptional activator takes part in the regulation of ICAM-1 expression during airway inflammation.

Intrapulmonary regulatory peptides come mainly from sensory nerve fibers and neuroendocrine cells in the lungs, which respond to challenges in the microenvironment. Intrapulmonary regulatory peptides exert important influence on the inflammation process and immune system, and probably influence all links of inflammation signal transduction including the activation of nuclear transcriptional factor, expression of adhesion molecules, and release of interleukins or other inflammatory media. Our previous studies indicated that vasoactive intestinal peptides (VIP) attenuated inflammation and injury in airway by way of upregulating enzyme synthesis against active oxygen[2]. We also demonstrated that epithelial growth factor (EGF) inhibited inflammatory adhesion[1]. It is reported that endothelin-1 (ET-1) increases inflammatory adhesion through modulating the expression of ICAM-1 or vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells or cardiac muscle cells[3]. In addition, ET-1 directly attracts monocytes as a chemotactic factor, stimulates the release of interleukins from PMNs, and enhances peroxide formation in PMNs irritated by F-Met-Leu-Phe (fMLP)[4]. It was also reported that calcitonin gene-related peptides (CGRP) increased adhesion during inflammation between endothelial cells and macrophages by way of stimulating the expression of adhesion molecules on endothelial cells[5]. Intrapulmonary regulatory peptides may play different modulatory roles in airway inflammation. The imbalance between these two kinds of regulatory peptides probably gives the explanation of the occurrence of the airway diseases.

For demonstrating the modulation and mechanism of different regulatory peptides on airway inflammation, the present experiments were carried out to testify the effects of regulatory peptides on NF-κB activation and ICAM-1 expression in BECs.

1 MATERIALS AND METHODS

1.1 Animals and reagents. New Zealand rabbits weighing 1.5-2 kg were used in the experiments. RPMI1640 culture medium, trypsin, VIP, EGF, ET-1, CGRP, and MG-132 were purchased from Sigma; Penicillin/ streptomycin and fetal bovine serum (FBS) were from Amresco; Anti-human ICAM-1 and IκB polyclonal antibody and immunocytochemistry kit were supplied by Zymed. Total RNA extract kit was supplied by Sigma; RT-PCR kit was purchased from Takara; α-32P-dCTP was supplied by Ya-Hui Co, Beijing. The O3 generator was supplied by the Nonferrous Metal Research Institute (Changsha, China).

1.2 BECs isolation and culture. BECs were isolated from New Zealand rabbits according to a method established by our lab[6]. Viability of the cells estimated by trypan blue staining was 88.6±2.2% and the purity of epithelial cells identified by Wright′s stain was 84.0±2.4%. BECs were resuspended to 106 cells/ml with RPMI1640 medium containing 5% FBS, streptomycin/penicillin. Then aliquots of cell resuspension were placed onto a collagen-coated 24-well culture plate and cultured at 37℃ in 5% CO2 in humidified atmosphere.

1.3 ICAM-1 analysis by immunocytochemistry. BECs were pretreated respectively with four kinds of peptides both in resting and O3-stressed condition. In resting condition BECs pretreated with VIP, EGF, ET-1 and CGRP were kept at 37℃ for 2 h, then were kept at room temperature for 2 h; while in O3-stressed condition BECs pretreated with VIP, EGF, ET-1 and CGRP were placed at 37℃ for 2 h, then were in 1.5 ppm O3 for 2 h before assay. Then the cells were centrifuged and sedimented on a glass slide which had been treated with polylysine. BECs were fixed in anhydrous alcohol, then treated with 3% H2O2. Non specific bindings were blocked by 10% goat serum. Incubation with the diluted specific primary antibody was performed at 37℃ for 1 h,then overnight at 4℃ in a moist chamber. After incubation with the corresponding diluted secondary antibody, sABC was added. The peroxidate activity was visualized by the DAB reactions. PBS replaced primary antibody or secondary antibody as a negative control.

1.4 ICAM-1 assay by RT-PCR. After various pretreatments mentioned above, total cellular RNA was extracted. RNA quality assayed by 1% agarose gel electrophoresis showed 5,18, 28 s three bands. OD260/OD280 of total RNA was 1.89±0.12. According to manufacturer instructions, after an initial reverse transcription at 50℃ for 30 min, samples were run for 25 cycles under the following conditions: 1 min denaturation at 94℃, 1 min annealing at 56℃,and 2 min extension at 72℃. RT-PCR products were assayed on 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Primers for ICAM-1 (5′-AGGTCCACCACTGACACGTT-3′ and 5′-CCAATATGATTCCACCCATG-3′) and GAPDH (5′-GAGCTGTTTGAGAACACCTC-3′ and 5′-TCACACTTCACTGTCACCTC-3′) yielded products of 300 bp and 600 bp respectively.

1.5 Preparation of nuclear extracts． After BECs were pretreated respectively for 15 min, the pretreatment was terminated by the addition of ice-cold PBS. The samples were resuspended in 400 μl buffers A (10 mmol/L HEPES, pH 7.8, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol, 0.5 mmol/L PMSF, 0.4 μg aprotinin) for 15 min on ice, then 25 μl 10% Nonidet P-40 was added, before the samples were vortexed for 30 s, and centrifuged at 10000 g for 1 min.The supernatant was discarded,and the cell pellet was resuspended in 100 μl buffer B (20 mmol/L HEPES pH 7.8, 0.4 mol/L KCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1 mmol/L EGTA, 1 mmol/L PMSF and 0.1 μg aprotinin). After being rocked vigorously at 10000 g for 15 min at 4℃, cellular debris were removed by centrifuging at 10000 g for 15 min at 4℃ and the supernatant fraction was stored at -70℃.

1.6 Measurement of NF-κB activity by EMSA． Nuclear extracts (5 μg) were incubated with 0.1 ng α-32P-dCTP-labeled double-stranded NF-κB sequence (5′-GAGATG ACG TAG TTT TCG CGC TT-3′). Optimal binding of NF-κB was obtained in buffer C (20 mmol/L HEPES pH 7.8, 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 0.5 mmol/L spermidine, 0.5 mmol/L spermine, 1 mmol/L dithiothreitol, 10% glycerol, 0.5 mmol/L PMSF, 0.1 μg aprotinin) at 4℃ for 30 min. Competitor assay was carried out by adding a 100-fold molar excess of unlabeled dsDNA. The reactions were electrophoresed on 5% polyacrylamide gel in 0.25 × TBE buffer. The gels were dried and autoradiographed.

1.7 Statistical analysis． The intensity and distribution of the specific immunocytochemical staining reaction were evaluated using a semiquantity method. Fifty cells every visual field were counted and 3-5 visual fields every slide was observed. The average photointensity values were obtained. DNA bands were measured with microscope graph analysis system. Calculation were performed with SPSS program. Kukey Kramer Multiple Comparison test were used to analyze group differences.The results were expressed as mean±SE. Significance was defined as a P value less than 0.05.

2 RESULTS

2.1 Effects of regulatory peptides on ICAM-1 protein expression

Immunocytochemistry showed that CGRP and ET-1 increased ICAM-1 protein expression in resting BECs; while VIP and EGF restrained ICAM-1 protein expression in O3-stressed BECs (Figs.1,2).

Fig.1.Effects of regulatory peptides on ICAM-1 protein expression in BECs (n=6). **P<0.01 vs control, #P<0.05 vs O3.

Fig.2.The ICAM-1 protein expression in BECs by immunocytochemistry. A: Resting BECs. B: ET-1. C: CGRP. D: O3. E: VIP+O3. F: EGF+O3.

2.2 Effects of regulatory peptides on ICAM-1 mRNA expression

RT-PCR results demonstrated that CGRP and ET-1 irritated ICAM-1 mRNA expression in resting BECs; VIP and EGF decreased ICAM-1 mRNA expression in O3-stressed BECs (Figs.3,4).

Fig.3.Effects of regulatory peptides on ICAM-1 mRNA expression in BECs (n=6). **P<0.01 vs control, #P<0.05 vs O3.

Fig.4.Effects of regulatory peptides on ICAM-1 mRNA expression in BECs by RT-PCR. M, DNA marker; 1, resting BECs; 2, ET-1; 3, CGRP; 4, O3; 5, VIP+O3; 6, EGF+O3.

2.3 Effects of MG-132 on ICAM-1 expression

NF-κB inhibitor MG-132 blocked ICAM-1 expression induced by O3, CGRP, and ET-1 (Figs.5-8).

2.4 Effects of regulatory peptides on activation of NF-κB

EMSA showed that an initial increase in NF-κB binding activity in O3-stressed BECs was observed at 5 min. Two peaks of NF-κB binding activity were ob-

Fig.5.Effects of MG-132 on ICAM-1 protein expression induced by O3, ET-1, and CGRP. A:O3; B:ET-1; C: CGRP; D: O3+MG-132; E: ET-1+MG-132; F: CGRP+MG-132.

Fig.6.Effects of MG-132 on ICAM-1 protein expression induced by O3, ET-1, and CGRP. *P<0.05 vs O3， #P<0.05 vs ET-1， +P<0.05 vs CGRP.

Fig.7.Effects of MG-132 on ICAM-1 mRNA expression induced by O3, ET-1, and CGRP. 1, O3; 2, O3+MG-132; 3, ET-1; 4, ET-1+MG-132; 5, CGRP; 6, CGRP+MG-132.

Fig.8.Effects of MG-132 on ICAM-1 mRNA expression induced by O3, ET-1, and CGRP (n=6). *P<0.05 vs O3， #P<0.05 vs ET-1； +P<0.05 vs CGRP．

served at 15 min and 1 h respectively, indicating that NF-κB was activated twice during 2 h in O3-stressed BECs. The retarded band was displaced by 100-fold molar excess of unlabeled NF-B probe. (Fig.9). CGRP and ET-1 promoted the activation of NF-κB in resting BECs; but VIP and EGF restrained the activation of the NF-κB in O3-stressed BECs (Fig.10).

Fig.9.Activity of NF-κB in O3-stressed BECs by EMSA． 1, 0 min; 2, 5 min; 3, 15 min; 4, 30 min; 5, 1 h; 6, 2 h; 7, 100-fold unlabeled probe.

Fig.10.Effects of regulatory peptides on activity of NF-κB in BECs by EMSA. 1, control; 2, ET-1; 3, CGRP; 4, O3; 5, VIP+O3; 6, EGF+O3.

3 DISCUSSION

ICAM-1 is constitutively expressed at low level in BECs and exerts an important effect in promoting inflammatory adhesion, promoting the tumor migration as well as adjusting body immune function[7]. ICAM-1 antibody blocked the adhesion in airway inflammation and blocked the development of airway inflammation effectively. ICAM-1 promoter harbors several cis-active regulatory sequences. Some elements have been identified that contribute to the regulation of the constitutive level of ICAM-1 gene transcription such as nuclear factor κB (NF-κB), signal transducer and activators of transcription (STAT)-1, STAT-3, SP-1, and Ets-Binding sites, etc[8]. Some experiments are carried out and find that ICAM-1 gene promoter has one alignment. The alignment can be distinguished by NF-κB/rel and AP-1, and it is necessary for interleukins and lipopolysaccharide to induce ICAM-1 expression on B lymphocytes and plasma cells[9].

By means of immunocytochemistry and RT-PCR we observed that O3 increased ICAM-1 protein expression and transcription. ET-1 and CGRP stimulated ICAM-1 expression in resting BECs, indicating that ET-1 and CGRP promoted inflammatory signal transduction and initialized the inflammation reaction in the airway; VIP and EGF decreased ICAM-1 expression in O3-stressed BECs, showing that VIP and EGF inhibited inflammatory signal transduction and abated the injury during inflammation. MG-132 blocked ICAM-1 protein expression and transcription in BECs induced by O3, ET-1 and CGRP, indicating that NF-κB modulated ICAM-1 transcription in BECs during airway inflammation. EMSA demonstrated that the respiratory tract diseases probably were related with the repeated activation of NF-κB. The reason of the repeated activation of NF-κB is not clear, but it is probable that different signal pathways are involved in the activation of NF-κB. CGRP and ET-1 promote the activation of NF-κB in resting BECs; VIP and EGF restrain the activation of NF-κB in O3-stressed BECs.

A conclusion can be drawn from the study that, through transcription of ICAM-1 and activation of NF-κB, BECs transmit the inflammatory or stress signals to the inflammatory cells and initiate the inflammatory reaction. VIP and EGF exerted a downregulatory effect to abate the injury while ET-1 and DGRP exerted an upregulatory effect to enhance the inflammation reaction. Over transcription of ICAM-1 in BECs, over activation of NF-κB, or abnormal distribution of regulatory peptides are possibly intrinsic factors of airway hyperresponsiveness.

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