Augmented levels of macrophage and Th1 cell-related cytokine mRNA in submandibular glands of MRL/lpr mice with autoimmune sialoadenitis

Abstract

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop destructive inflammation of the salivary and lachrymal glands resembling Sjögren's syndrome (SS), representing an animal model to study this disease. We used in situ hybridization with synthetic radiolabelled oligonucleotide probes to examine expression of mRNA encoding pro- and anti-inflammatory cytokines in submandibular glands of 2, 3, 4 and 5-month-old MRL/lpr mice. Phenotypic composition of submandibular gland infiltrates was evaluated by immunohistochemistry. Cells expressing tumour necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and IL-12 mRNA were strongly up-regulated at about the time of onset of sialoadenitis, suggesting a role of these cytokines in development of the disease. Interferon-gamma (IFN-gamma) and cytolysin mRNA-expressing cells were gradually up-regulated over the disease course up to 5 months of age, the time when sialoadenitis is at its height, favouring a role of these cytokines in progression of the disease as well. Low levels of IL-10 and transforming growth factor-beta (TGF-beta) mRNA-expressing cells were observed at 2, 3 and 4 months of age, and were almost undetectable at 5 months. Maximum levels of CD4+, CD8+ and interdigitating/dendritic cells, as well as of MHC class II and MHC class I expression were seen at 3 months, with CD4+ outnumbering CD8+ cells. Maximum levels of macrophages were seen at 4 months of age. These data argue for a major role of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, IL- 12, IFN-gamma and cytolysin in initiation and perpetuation of autoimmune sialoadenitis in MRL/lpr mice, probably in conjunction with an insufficiency of the anti-inflammatory cytokines TGF-beta and IL-10.

Fig. 1

Autoradiograms of in situ hybridization. A cluster of grains over individual cells show: (a) transforming growth factor-alpha mRNA-expressing cell in the submandibular gland inflammatory infiltrate in 2-month-old MRL/lpr mouse (×400), and (b) IL-6 mRNA-expressing cell among spleen mononuclear cells in 5-month-old MRL/lpr mouse (×400).

Fig. 2

The histological appearance of the submandibular glands of MRL/lpr mice. (a) Mild mononuclear cell infiltration in 2-month-old mouse (×200), and (b) severe mononuclear cell infiltration in 5-month-old mouse (×200).

Fig. 3

Numbers of tumour necrosis factor-alpha (TNF-α) mRNA-expressing cells, detected by in situ hybridization with ³⁵S-labelled synthetic oligonucleotide probes, per 100 mm² submandibular gland tissue sections (a), and per 10⁵ spleen mononuclear cells (MNC) (b), of MRL/lpr mice and BALB/c mice at 2, 3, 4 and 5 months of age. Each column indicates mean value of five mice and bars indicate s.d. Levels of significance for differences between MRL/lpr mice and age-matched control BALB/c mice: *P < 0.05; **P < 0.01; ***P < 0.001. There were significantly higher numbers of TNF-α mRNA-expressing cells both in submandibular glands and among spleen MNC in MRL/lpr mice compared with control BALB/c mice at 3, 4 and 5 months of age. Peak numbers of submandibular gland TNF-α mRNA-expressing cells occurred at 3 months of age, while peak numbers of TNF-α mRNA-expressing spleen MNC were registered at 4 months of age.

Fig. 4

Numbers of IL-1β mRNA-expressing cells in submandibular glands (a) and among spleen mononuclear cells (MNC) (b) of MRL/lpr mice and control BALB/c mice. For explanation see legend to Fig. 3.

Fig. 5

Numbers of IL-6 mRNA-expressing cells in submandibular glands (a) and among spleen mononuclear cells (MNC) (b) of MRL/lpr mice and control BALB/c mice. For explanation see legend to Fig. 3.

Fig. 6

Numbers of IFN-γ mRNA-expressing cells in submandibular glands (a) and among spleen mononuclear cells (MNC) (b) of MRL/lpr mice and control BALB/c mice. For explanation see legend to Fig. 3. The numbers of IFN-γ mRNA-expressing cells of MRL/lpr mice at 2, 3, 4 and 5 months of age were significantly higher compared with age-matched BALB/c mice, where IFN-γ mRNA expression was not detectable.

Fig. 7

Numbers of cytolysin mRNA-expressing cells in submandibular glands (a) and among spleen mononuclear cells (MNC) (b) of MRL/lpr mice and control BALB/c mice. For explanation see legend to Fig. 3.

Fig. 8

Numbers of IL-12 mRNA-expressing cells in submandibular glands of MRL/lpr mice and control BALB/c mice. For explanation see legend to Fig. 3.