INCREASED INTERLEUKIN-1 RECEPTOR IMMUNOREACTIVITY IN SYMPATHETIC NEURONS IN GUINEA-PIG COELIAC GANGLIA INCUBATED IN VITRO

Judy L. Morris, Pat Vilimas, Keiko Mayne, Phillip Jobling, Toshihiko Shimizu, Ian L. Gibbins, Centre for Neuroscience, Dept Anatomy & Histology, Flinders University, GPO Box 2100, Adelaide, SA.

Pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF), interleukin-1beta (IL1B) and interleukin-6 (IL6) are upregulated in inflammatory conditions including bacterial infection of the abdomen. Severe inflammation leads to disturbances in splanchnic blood flow, and sometimes to uncontrolled hypotension and endotoxic shock. Cytokines can alter excitability of enteric neurons and transmission from sympathetic neurons in the gut wall. However, it is not known whether sympathetic neurons regulating gastrointestinal blood flow, a major determinant of arterial blood pressure, also are targets of pro-inflammatory cytokines. We have identified potential sites of action of IL1B on neurons regulating gastrointestinal blood flow using antisera raised against the C-terminus or the N-terminus of the interleukin-1 receptor type I (IL1RI), the membrane-bound receptor mediating actions of IL1B. Guinea-pigs were killed by stunning and exsanguination, and coeliac ganglia were removed and fixed in Zamboni's solution prior to processing for multiple-labelling immunohistochemistry or were frozen in liquid nitrogen prior to protein extraction and Western blotting. Some ganglia were incubated for 2 to 4h at 37^oC in HEPES-buffered balanced salt solution, with or without TNF, IL1B or IL6 (200ng/ml), prior to fixation or freezing. IL1RI-immunoreactivity (IR) was present in 8% of neuron somata in untreated preparations. The proportion of neurons with IL1RI-IR increased significantly after incubation for 4h without cytokine (16%) or with TNF or IL1B (16% and 18%, respectively). However, the proportion of neurons with IL1RI-IR was increased by 2h incubation in IL6 (16%), and increased further by 4h (26%). These increases occurred predominantly in neuropeptide Y-IR, vasoconstrictor neurons. Western blotting demonstrated an IL1RI-IR protein band of approximately 70kDa that was more dense after in vitro incubation than in freshly-frozen ganglia. This IL1RI-IR is likely to represent membrane-bound receptors for interleukin-1beta on coeliac ganglion neurons, which may modulate sympathetic regulation of intestinal blood flow during inflammation or injury.