德国小蠊提取物可增加支气管上皮细胞白细胞介素-8的表达。

German cockroach extract increases bronchial epithelial cell interleukin-8 expression.

作者信息

Bhat R K, Page K, Tan A, Hershenson M B

机构信息

Department of Pediatrics, University of Chicago, Chicago, Illinois, USA.

出版信息

Clin Exp Allergy. 2003 Jan;33(1):35-42. doi: 10.1046/j.1365-2222.2002.01481.x.

DOI:10.1046/j.1365-2222.2002.01481.x

PMID:12534547

Abstract

BACKGROUND

Cockroach exposure has been recognized as a common trigger for asthma. While dust mite and Aspergillus fumigatus aeroallergens have been noted to have direct effects on airway epithelium, direct effects of cockroach proteins have not been determined.

OBJECTIVE

The purpose of this study was to investigate whether cockroach extract has a direct pro-inflammatory effect on airway epithelium.

METHODS

We examined the effect of crude German cockroach (Blattella germanica) extract on IL-8 expression in a human bronchial epithelial cell line (16HBE14o-cells) and primary human bronchial epithelial cells. Transcription from the IL-8 promoter and protein abundance were determined by reporter assay and enzyme-linked immunosorbent assay (ELISA), respectively. Endotoxin levels in the crude cockroach extracts were determined using the Limulus Amebocyte Lysate assay. Protease activity was assessed using Azocoll as a substrate.

RESULTS

We found that crude cockroach extract induced a synergistic increase in TNF-alpha-induced transcription from the IL-8 promoter. The synergistic effect was observed with as little as 0.3 micro g/mL of crude cockroach extract, while larger concentrations (30 micro g/mL) approximately doubled TNF-alpha-induced IL-8 promoter activity. Similar effects of cockroach extract on IL-8 protein abundance were observed in both 16HBE14o- and primary human bronchial epithelial cells. Addition of endotoxin at concentrations found in the cockroach extract had no effect on TNF-alpha-mediated IL-8 expression. The serine protease inhibitors aprotinin and phenylmethylsulphonyl fluoride abolished cockroach-induced synergy, while the cysteine protease inhibitors E64 and leupeptin had little effect. Measurement of protease activity using Azocoll as a substrate confirmed the presence of protease activity in cockroach extracts. Addition of recombinant Bla g 2, Bla g 3 and Bla g 5 had no effect on TNF-alpha-induced IL-8 promoter activation. Finally, cockroach extract also increased TNF-alpha-induced transcription from the IL-6 promoter.

CONCLUSIONS

German cockroach extract contains novel serine protease activity which has a direct pro-inflammatory effect on airway epithelial cells

摘要

背景

接触蟑螂已被公认为哮喘的常见触发因素。虽然已注意到尘螨和烟曲霉气传变应原对气道上皮有直接影响，但蟑螂蛋白的直接影响尚未确定。

目的

本研究旨在调查蟑螂提取物是否对气道上皮有直接促炎作用。

方法

我们检测了德国小蠊粗提物对人支气管上皮细胞系（16HBE14o-细胞）和原代人支气管上皮细胞中白细胞介素-8（IL-8）表达的影响。分别通过报告基因检测和酶联免疫吸附测定（ELISA）确定IL-8启动子的转录和蛋白丰度。使用鲎试剂法测定蟑螂粗提物中的内毒素水平。以偶氮酪蛋白为底物评估蛋白酶活性。

结果

我们发现蟑螂粗提物可协同增强肿瘤坏死因子-α（TNF-α）诱导的IL-8启动子转录。低至0.3μg/mL的蟑螂粗提物即可观察到协同作用，而更高浓度（30μg/mL）可使TNF-α诱导的IL-8启动子活性增加约一倍。在16HBE14o-细胞和原代人支气管上皮细胞中均观察到蟑螂提取物对IL-8蛋白丰度有类似影响。添加蟑螂提取物中所含浓度的内毒素对TNF-α介导的IL-8表达无影响。丝氨酸蛋白酶抑制剂抑肽酶和苯甲基磺酰氟可消除蟑螂诱导的协同作用，而半胱氨酸蛋白酶抑制剂E64和亮抑酶肽作用甚微。以偶氮酪蛋白为底物测定蛋白酶活性证实蟑螂提取物中存在蛋白酶活性。添加重组变应原Bla g 2、Bla g 3和Bla g 5对TNF-α诱导的IL-8启动子激活无影响。最后，蟑螂提取物还增强了TNF-α诱导的IL-6启动子转录。