Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC316

Poster Communications

Cytokine regulation of natural antimicrobial peptide expression in cultured human vaginal epithelial and endocervical cells

D. Abbott1, E. C. Chin-Smith1, C. M. Foster2, A. H. Shennan1, R. M. Tribe1

1. Division of Womens Health, School of Medicine, King's College London and King's Health Partners, London, United Kingdom. 2. Faculty of Medicine, Nursing and Health Sciences, Monash Univeristy, Melbourne, Victoria, Australia.

Introduction: Natural antimicrobial peptides (NAPs) have been identified, in the female reproductive tract (King et al., 2007) and in cervico-vaginal fluid (Stock et al., 2009), as an integral part of innate immune defence. The role of NAPs in spontaneous preterm birth is not well described. Based on data obtained in vivo (Chandiramani et al., 2010), we hypothesised that granulocyte-macrophage colony-stimulating factor (GMCSF) and other pro-inflammatory cytokines (an early indicator of inflammation and risk of preterm birth) would regulate expression of NAPs in reproductive tract epithelia. Our aim was to assess impact of interleukin-1beta (IL-1β) and GMCSF on elafin, secretory leucocyte protease inhibitor (SLPI) and human-beta defensin-2 (HBD-2) mRNA expression in vaginal epithelial (VK2/E6E7) and endocervical (END-1) cells. Methods: VK2/E6E7 (VK2) and END-1 cells (80% confluence) were treated with IL-1β and GMCSF (1 ng/ml and 10 ng/ml) for 6 and 24 h. Elafin, SLPI and HBD-2 mRNA expression, relative to stably expressed GAPDH (VK2 cells) or a normalisation factor of 3 housekeeper genes (END-1 cells), was assessed by quantitative real-time PCR. Data are expressed as mean (±SE) and analysed using analysis of variance with repeated measures and a Bonferroni’s multiple comparison post-hoc test or Freidman’s test with Dunn’s post-hoc analysis. Experiments were performed in triplicate for n=6 passages for VK2 cells and n=3 for END-1 cells. Results: Elafin, SLPI and HBD-2 transcripts are present in VK2 and END-1 cells. In VK2 cells, IL-1β (10 ng/ml) significantly increased elafin mRNA expression (14.28 ± 4.42 vs control 3.15 ± 1.62, n=6, p<0.001), SLPI (7.91 ± 3.60 vs control 0.68 ±0.09, n=6, p<0.05) and HBD-2 (0.016 ± 0.0052 vs control 0.001 ±0.0004, n=6, p<0.001). GMCSF (10 ng/ml) significantly inhibited elafin in VK2 cells (2.53 ±0.68 vs control 6.43 ±1.92, n=6, p<0.05). In END-1 cells, IL-1β (1 ng/ml) significantly increased elafin mRNA expression (146260 ±24314 vs control 77388 ±22017, n=3, p< 0.0001) and HBD-2 (ILβ 10 ng/ml) (48.47 ±10.15 vs control 9.06 ±2.92, n=3, p=0.007). GMCSF (1 ng/ml) significantly inhibited elafin expression in END-1 cells; (19631 ±4697 vs control, 29896 ±7300, n=3, p=0.02) and SLPI (10 ng/ml)(26758 ±3741 vs control 38458 ±3278,n=3, p=0.01). GMCSF had no effect on HBD-2 expression in either cell line. Conclusions: This study provides valuable information on the regulation of NAPs by inflammatory cytokines in vaginal epithelial and endocervical cell lines and is the first to demonstrate the effect of GMCSF. GMCSF suppression of elafin and SLPI provides a novel mechanism that may explain impaired immune responses against ascending infection in women at risk of preterm birth and warrants further investigation. Funding: Tommy’s Charity & Wellbeing of Women

Where applicable, experiments conform with Society ethical requirements