Vasoactive intestinal peptide inhibits TNF-α-induced apoptotic events in acinar cells from nonobese diabetic mice submandibular glands

Mario Calafat,Nicolás Pregi,Claudia Pérez Leirós,Alcira Nesse,Valeria Roca,Luciana Larocca,Vanesa Hauk

doi:10.1186/ar2671

Abstract

IntroductionThe role of apoptotic secretory epithelium as a pro-inflammatory triggering factor of exocrine dysfunction is currently explored in Sjogren's syndrome patients and in the nonobese diabetic (NOD) mouse model. Vasoactive intestinal peptide (VIP) has anti-inflammatory effects in various models of chronic inflammation. Our goal was to analyse the effect of TNF-α on apoptotic mediators in isolated acinar cells from NOD submandibular gland and their modulation by VIP.MethodsAcinar cells were isolated from submandibular glands of 16-week-old NOD females with salivary flow decline. Age-matched BALB/c females or eight-week-old NOD females were used as controls. Apoptotic mediators and TNF-α receptor expression were assessed by immunoblotting and RT-PCR, caspase 3 activity was assessed by optical density at 405 nm with Ac-DEVD-pNA as a substrate and chromatin condensation by Hoechst stain. They were evaluated in resting conditions and after a 3.5 or 6 hours of culture with TNF-α. VIP effects in acinar cells were assessed at 100 nM in TNF-α-treated cultures and VIP receptor functional assays by radio immunoassay (cAMP) or enzymatic detection (amylase).ResultsNOD acinar cells at 16 weeks present an increased expression of TNF-α receptor1 together with increased Bax, tumour protein 53-induced nuclear protein1α (TP53INP1α), caspase 3 activity and chromatin condensation. Acini from NOD mice were more sensitive to TNF-α-induced increases of apoptotic mediators than control cells. VIP inhibited TNF-α-induced apoptotic events through functional VPAC1 receptors coupled to the protein kinase A (PKA) signalling pathway.ConclusionsOur results indicate that acinar cells isolated from submandibular glands of NOD mice with salivary dysfunction are more sensitive to apoptosis induced by TNF-α which could be prevented by VIP through a PKA-mediated pathway.

Highlights

The role of apoptotic secretory epithelium as a pro-inflammatory triggering factor of exocrine dysfunction is currently explored in Sjogren's syndrome patients and in the nonobese diabetic (NOD) mouse model
Our results indicate that acinar cells isolated from submandibular glands of non obese diabetic (NOD) mice with salivary dysfunction are more sensitive to apoptosis induced by TNF-α which could be prevented by Vasoactive intestinal peptide (VIP) through a protein kinase A (PKA)-mediated pathway
Observations that acinar cells may be actively involved in the pathogenesis of Sjögren's syndrome encourage the search for molecules that could mediate these processes, which could even arise as biomarkers for diagnosis and/or disbp: base pair; FBS: fetal bovine serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IL: interleukin; NOD: non obese diabetic; NOS: nitric oxide synthase; PBS: phosphate-buffered saline; PKA: protein kinase A; RT-PCR: reverse transcription polymerase chain reaction; TNF-α: tumour necrosis factor-alpha; TNF-α-R: tumour necrosis factor-alpha-receptor; TP53INP1: tumour protein 53-induced nuclear protein 1; terminal UTP nucleotide end-labelling method (TUNEL): terminal UTP nucleotide end labelling method; VIP: vasoactive intestinal peptide

Summary

Introduction

The role of apoptotic secretory epithelium as a pro-inflammatory triggering factor of exocrine dysfunction is currently explored in Sjogren's syndrome patients and in the nonobese diabetic (NOD) mouse model. Observations that acinar cells may be actively involved in the pathogenesis of Sjögren's syndrome encourage the search for molecules that could mediate these processes, which could even arise as biomarkers for diagnosis and/or disbp: base pair; FBS: fetal bovine serum; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IL: interleukin; NOD: non obese diabetic; NOS: nitric oxide synthase; PBS: phosphate-buffered saline; PKA: protein kinase A; RT-PCR: reverse transcription polymerase chain reaction; TNF-α: tumour necrosis factor-alpha; TNF-α-R: tumour necrosis factor-alpha-receptor; TP53INP1: tumour protein 53-induced nuclear protein 1; TUNEL: terminal UTP nucleotide end labelling method; VIP: vasoactive intestinal peptide. The NOD mouse model of Sjögren's syndrome, at the pre-diabetic stage, has the unique characteristic of developing a deep secretory dysfunction with mild infiltration of the glands [7] consistent with a structural-dysfunctional aetiology

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