The crux of positive controls - Pro-inflammatory responses in lung cell models

Positive controls are an important feature in experimental studies as they show the responsiveness of the model under investigation. An often applied reagent for a pro-inflammatory stimulus is the endotoxin lipopolysaccharide (LPS), which has been shown to induce a cytokine release by various cell cultures. The effect of LPS in monocultures of 16HBE14o-, a bronchial cell line, and of A549, an alveolar cell line, were compared in submerged and air-liquid interface cultures, as well as in co-cultures of the two epithelial cells with monocyte-derived macrophages and dendritic cells. The protein and mRNA levels of the two most relevant pro-inflammatory mediators, Tumor necrosis factor alpha (TNF) and Interleukin 8 (CXCL8), were measured after 4 h and 24 h exposure. 16HBE14o- cells alone as well as in co-cultures are non-responsive to an LPS stimulus, but an already increased basal expression of both pro-inflammatory mediators after prolonged time in culture was observed. In contrary, A549 in monocultures showed increased CXCL8 production at the gene and protein level after LPS exposure, while TNF-levels were below detection limit. In A549 co-cultured with immune cells both mediators were upregulated. This study shows the importance of a careful evaluation of the culture system used, including the application of positive controls. In addition, the use of co-cultures with immune cells more adequately reflects the inflammatory response upon exposure to toxicants.