Budesonide enhances Toll-like receptor 2 expression in activated bronchial epithelial cells

Abstract

Endotoxin, tumor necrosis factor (TNF), and organic dust constitute proinflammatory stimuli involved in the initiation of inflammation. The major receptor for endotoxin (lipopolysaccharide [LPS]) is Toll-like receptor 4 (TLR4), whereas TLR2 binds to agents from gram-positive bacteria. The aim of the study was to elucidate whether TLR2 and TLR4, expressed on primary bronchial epithelial cells (PBECs), are influenced by exogenous (organic dust and LPS) and endogenous (TNF) stimuli and whether this interaction is influenced by a glucocorticosteroid. The cells were exposed to LPS (10 µg/ml), TNF (10 ng/ml), or dust (100 µg/ml) 1.5 and 6 h, in the presence or absence of budesonide (10⁻⁶ M) in vitro. The mRNA expression of interleukin (IL)-6, IL-8, TLR2, and TLR4 were measured with real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and IL-6 and IL-8 release was assessed with enzyme-linked immunosorbent assay (ELISA). To elucidate the importance of TLR-signaling for IL-6 and IL-8 secretion, the effect of TLR-blockers was studied. Endotoxin, TNF, and dust stimulated the release of IL-6 and IL-8 in a time-dependent manner. Budesonide significantly attenuated the release and expression of IL-6 and IL-8 after exposure. Budesonide did not influence TLR expression, but costimulation with LPS, TNF, or dust together with budesonide increased TLR2 expression synergistically. Blocking of TLR2 and TLR4 reduced cytokine secretion in stimulated cells. Budesonide reduced IL-6 and IL-8 production and enhanced expression of TLR2 in PBECs only in the presence of a proinflammatory stimulus. These findings contribute to our understanding of the beneficial effects of glucocorticosteroids during chronic obstructive pulmonary disease (COPD) exacerbations and asthma, which are frequently caused by microorganisms.

Keywords::

• Glucocorticoids
• inflammation
• innate immunity
• Toll-like receptor (TLR)

Acknowledgments

The authors would like to thank Dr. Karin Strandberg for the PBECs and Karin Sahlander for valuable FACS knowledge (The Institute of Environmental Medicine, Lung and allergy research), and Dr. Lotta Orre (Thoracic Surgery Clinic) and Dr. Anders Höög (Department of Pathology and Cytology), Karolinska University Hospital, for help with providing human lung tissue.

Declaration of interest

The study was supported by grants from Swedish Heart and Lung Foundation, Karolinska Institutet, and Swedish Farmer Foundation for Agriculture Research. Kjell Larsson has, during the last five years, on one or more occasion served in an advisory board and/or served as speaker and/or participated in education arranged by AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, MSD, Nycomed and Pfizer. Kjell Larsson has also received unrestricted research grants from Boehringer Ingelheim, GlaxoSmithKline, and AstraZeneca during the last five years.