Efficient IL-18 and IL-18BPc adenoviral gene transfer to cultured murine submandibular gland epithelial cells and successful murine retrograde submandibular duct cannulation to modulate IL-18 function in the salivary gland of animal models of Sjogren's syndrome
https://doi.org/10.1186/ar1615
© BioMed Central Ltd 2005
Received: 11 January 2005
Published: 17 February 2005
Keywords
Introduction
We recently demonstrated that the proinflammatory and immuno-regulatory cytokine IL-18 is strongly upregulated in salivary glands (SG) of Sjogren's syndrome (SS) patients. IL-18 gain or loss of function experiments in animal models of human diseases have provided evidence of the pathogenic role of this cytokine in chronic inflammation. However, in SS the pathogenic relevance of IL-18 in the development of murine autoimmune sialoadenitis has not as yet been evaluated. Here we describe in vitro experiments in which murine SG epithelial cells were tested for transfection efficiency with IL-18 and IL-18BPcAdV gene transfer. In addition, in preparation for in vivo SG modulation of IL-18 function by AdV-mediated gene transfer, we optimised retrograde SG cannulation in mice.
Methods
Primary SG ductal epithelial cells (SG-DEC), established from submandibular glands of C57BL/6 mice using the explant outgrowth technique, as well as a murine SG-DEC line were used for AdV transfection. A mIL-18AdV from plasmid IL-18 TG3652 [1] was developed in our laboratory while we used the same mIL-18BPcAdV construct as previously reported [2]. AdVs encoding for LacZ or luciferase were used as controls. All AdVs were incubated at 50 MOI. Efficiency of gene transfer was evaluated through detection of IL-18 and IL-18BPc protein expression assessed by both immunostaining and western blot. Beta-galactosidase staining was performed to evaluate efficiency of transfection of LacZAdV. Feasibility of local delivery of compounds through retrograde cannulation of murine submandibular glands was also tested.
Results
(a) Expression of murine IL-18 in salivary gland ductal epithelial cells (SG-DEC) transfected with IL-18AdV but not with LacZAdV (b). (c) Beta-galactosidase staining confirmed effective transfection of SG-DEC by LacZAdV.
(a) Expression of murine IL-18BPc in salivary gland ductal epithelial cells transfected with IL-18BPcAdV but not with LucAdV (b) or medium alone (c).
Conclusion
Here we report for the first time evidence of high and sustained efficiency of IL-18 and IL-18BPc AdV gene transfer in murine SG-DEC. In addition, we successfully adapted a cannulation technique previously used in bigger animals for in vivo local delivery of modulatory molecules to murine salivary glands.
Local delivery of IL-18/IL-18BPc adenoviral vectors in vivo in salivary glands of NOD mice and other murine models of SS through retrograde submandibular excretory duct cannulation will provide evidence of a possible pathogenic role of IL-18 in participating in autoimmune sialoadenitis and will establish a rationale for using IL-18 blocking agents as therapeutic tools in SS.
Authors’ Affiliations
References
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