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Mast cells, smooth muscle and inflammation in asthma
S124 RAGE is expressed by human airway smooth muscle cells and expression is increased in asthma
  1. L Di Candia,
  2. E Gomez,
  3. J Challiss,
  4. C E Brightling,
  5. R Saunders
  1. University of Leicester, Leicester, UK

Abstract

Background Airway smooth muscle (ASM) dysfunction contributes to the airway hyper-responsiveness and airflow obstruction observed in asthma. The receptor for advanced glycosylation end products (RAGE) is a pattern recognition receptor activated by endogenous danger signals, including high mobility group box 1 (HMGB1). RAGE and HMGB1 are implicated in many pathophysiological states, such as diabetes and Alzheimer's disease. RAGE is abundantly expressed in healthy adult lung, where it might serve a homeostatic function, for example, in alveolar type I epithelial cells; however, RAGE expression/function in other airway cell-types is poorly characterised. We hypothesised that changes in RAGE and HMGB1 expression may contribute to ASM dysfunction in asthma. Therefore, RAGE/HMGB1 expression was investigated in human primary ASM cells.

Methods ASM was microdissected from bronchial biopsies and large airway specimens obtained at lung resection surgery. Ex-vivo ASM cells were characterised for a-smooth muscle actin and used between passages 2–5. Cells were serum deprived in ITS medium for 72-h prior to experimentation. RAGE and HMGB1 mRNA expression was measured using RT-PCR and qRT-PCR, and protein expression by western blotting, immunofluorescence and flow cytometry.

Results ASM cells were shown to express the full-length and three soluble RAGE transcripts by RT-PCR (n=5), and an HMGB1 transcript by qRT-PCR (n=6). By flow cytometry membrane-localised RAGE expression was shown to be significantly increased in ASM cells isolated from asthmatics (18.0±5.4% of the ASM population expressed RAGE, n=6) vs non-asthmatics (8.6±6.5%, n=7; *p<0.05), and a trend towards decreased HMGB1 expression in asthmatics (18.8±10.5%, n=6) vs non-asthmatics (35.7±19.1%, n=6) was observed; however, these values were not significantly different. RAGE and HMGB1 expression were confirmed by immunofluorescence and western blotting.

Conclusion Human ASM cells express RAGE and its ligand HMGB1 at the mRNA and protein levels. Membrane-localised RAGE protein expression is significantly increased and there is a trend towards a decrease in HMGB1 protein expression in ASM isolated from asthmatic vs non-asthmatic subjects. The contribution of these changes to ASM dysfunction in asthma requires further investigation.

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