Defective signalling in salivary glands precedes the autoimmune response in the non-obese diabetic mouse model of sialadenitis

Abstract

The spontaneous non-obese diabetic (NOD) mouse model of Sjögren's syndrome provides a valuable tool to study the onset and progression of both the autoimmune response and secretory dysfunction. Our purpose was to analyse the temporal decline of salivary secretion in NOD mice in relation to the autoimmune response and alterations in various signalling pathways involved in saliva secretion within each salivary gland. A progressive loss of nitric oxide synthase activity in submandibular and parotid glands started at 12 weeks of age and paralleled the decline in salivary secretion. This defect was associated with a lower response to vasoactive intestinal peptide in salivary flow rate, cAMP and nitric oxide/cGMP production. No signs of mononuclear infiltrates or local cytokine production were detectable in salivary glands in the time period studied (10-16 weeks of age). Our data support a disease model for sialadenitis in NOD mice in which the early stages are characterized by defective neurotransmitter-mediated signalling in major salivary glands that precedes the autoimmune response.

Fig. 1

Progressive decrease of nitric oxide synthase and vascular endothelial peptide (VIP)-activated signalling through NO/cGMP in non-obese diabetic (NOD) mice glands. (a) Basal NOS activity was determined in submandibular and parotid glands of NOD mice at different ages from 10 to 20 weeks and glands of BALB/c mice of the ages indicated (insert) as described in Materials and methods. Values represent the mean ± s.e. of at least six different glands. *P < 0·05 versus basal of the corresponding gland from 10-week-old NOD mice. (b) The effect of VIP on NOS activity (10 n M VIP, upper panel) and cGMP accumulation (100 n M VIP, lower panel) was assessed in submandibular or parotid glands of 10-, 14- and 16-week-old NOD mice and BALB/c mice of 16 weeks (insert). Each value represents the mean ± s.e. of at least four determinations. *P < 0·05 versus basal of the corresponding gland at the same age.

Fig. 2

Histological studies of non-obese diabetic (NOD) salivary glands. Submandibular and parotid slices of NOD mice of 10, 14 and 16 weeks of age and BALB/c mice of 16 weeks were fixed and stained as described in Materials and methods. Duct number was determined by counting ducts in a survey of 20 fields and results shown are means ± s.e. of at least six different gland slices. The presence of mononuclear infiltrates was monitored in the whole slice directly and after anti-CD3 immunostaining, the ratios shown indicate the number of gland slices containing at least one infiltrate out of the total number of corresponding glands analysed. Sections shown are representative of six to nine other glands analysed similarly; the arrow indicates picnotic nuclei, bar = 10 µm..

Fig. 3

Circulating autoantibodies against salivary gland proteins. (a) Submandibular (SM) and parotid (P) glands of BALB/c mice were homogenized and fractionated on 10% sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE). Proteins transferred to nitrocellulose membranes were incubated with 1/100 dilutions of sera from BALB/c or non-obese diabetic (NOD) mice of the ages indicated and revealed as described in Materials and methods. (b) Fractionated proteins from submandibular (SM) and parotid (P) glands of NOD mice of the ages indicated obtained as in (a) were incubated with 1/100 dilution of the sera from the same NOD mice and revealed as described in Materials and methods. Each blot shown is representative of three others assayed similarly; the arrowhead indicates relevant proteins revealed by NOD sera at 16 weeks of age.