German cockroach extract increases bronchial epithelial cell interleukin‐8 expression

Summary 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-α-induced transcription from the IL-8 promoter. The synergistic effect was observed with as little as 0.3 µg/mL of crude cockroach extract, while larger concentrations (30 µg/mL) approximately doubled TNF-α-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-α-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-α-induced IL-8 promoter activation. Finally, cockroach extract also increased TNF-α-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