Splenic nerve is required for cholinergic antiinflammatory pathway control of TNF in endotoxemia

Abstract

The autonomic nervous system maintains homeostasis through its sympathetic and parasympathetic divisions. During infection, cells of the immune system release cytokines and other mediators that cause fever, hypotension, and tissue injury. Although the effect of cytokines on the nervous system has been known for decades, only recently has it become evident that the autonomic nervous system, in turn, regulates cytokine production through neural pathways. We have previously shown that efferent vagus nerve signals regulate cytokine production through the nicotinic acetylcholine receptor subunit alpha7, a mechanism termed "the cholinergic antiinflammatory pathway." Here, we show that vagus nerve stimulation during endotoxemia specifically attenuates TNF production by spleen macrophages in the red pulp and the marginal zone. Administration of nicotine, a pharmacological agonist of alpha7, attenuated TNF immunoreactivity in these specific macrophage subpopulations. Synaptophysin-positive nerve endings were observed in close apposition to red pulp macrophages, but they do not express choline acetyltransferase or vesicular acetylcholine transporter. Surgical ablation of the splenic nerve and catecholamine depletion by reserpine indicate that these nerves are catecholaminergic and are required for functional inhibition of TNF production by vagus nerve stimulation. Thus, the cholinergic antiinflammatory pathway regulates TNF production in discrete macrophage populations via two serially connected neurons: one preganglionic, originating in the dorsal motor nucleus of the vagus nerve, and the second postganglionic, originating in the celiac-superior mesenteric plexus, and projecting in the splenic nerve.

Conflict of interest statement

Conflict of interest statement: K.J.T. and M.R.-B. are inventors on technology related to the topic.

Figures

Fig. 1.

Anatomical distribution of spleen TNF in endotoxemia. Mice received 10 mg/kg LPS i.p. and were killed 10, 30, 60, 90, or 120 min later. (A) TNF was not detectable at 10 min. (B) At 30 min, TNF immunoreactivity was observed in the MZ, a thin rim of specialized cells surrounding the white pulp (WP), and in the red pulp (RP). (C) At 60 min, the number of TNF-positive cells increased in the MZ and the RP. (D) At 2 h, TNF was observed in cells of the WP. (Magnification: ×100.) (E and F) TNF protein content in the spleen peaked at 60 min (E) and correlated with serum TNF concentration (F). (G) High-power magnification showing TNF within spleen cells (arrowhead). (Magnification: ×630.) Pictures are representative of at least five mice per group and three to four sections per mouse.

Fig. 2.

Spleen macrophages costain with TNF in endotoxemia. Spleens harvested 60 min after LPS administration were stained for TNF (red) and markers for macrophage subpopulations (green). (A–F) TNF costained with (arrowheads) marginal metallophilic macrophages (MMM) (A and B), MZ macrophages (MZM) (C and D), and RP macrophages (RPM) (E and F). (G and H) Neutrophils, Gr1-positive cells, did not costain with TNF. (Magnification: A, C, E, and G, ×100; B, D, F, and H, ×400.) MZ, marginal zone; WP, white pulp; RP, red pulp.

Fig. 3.

Vagus nerve stimulation attenuates spleen TNF immunoreactivity in endotoxemia. (A–D) Rats were subjected to vagus nerve stimulation (C and D) or sham surgery (A and B). Spleens were harvested 60 min after LPS. In sham animals, TNF was detected in the MZ and RP. Vagus nerve stimulation significantly decreased TNF immunoreactivity throughout the spleen. (Magnification: A and C, ×25; C and D, ×100.) MZ, marginal zone; WP, white pulp; RP, red pulp.

Fig. 4.

Nicotine attenuates spleen TNF immunoreactivity in endotoxemia. Mice were treated with 2 mg/kg nicotine i.p. 30 min before 10 mg/kg LPS i.p. Spleens were harvested 60 min after LPS administration and stained for TNF. (A–D) Spleens of nicotine-treated mice (C and D) showed attenuated TNF immunoreactivity in the MZ and RP compared with spleens from PBS-treated mice (A and B). (Magnification: A and C, ×25; B and D, ×100.) MZ, marginal zone; WP, white pulp; RP, red pulp.

Fig. 5.

Nerve endings terminate adjacent to TNF-producing macrophages in spleen. Rat spleens harvested 60 min after 15 mg/kg LPS i.p. were stained for the synaptic vesicle protein synaptophysin, TNF, and macrophages. Synaptophysin-positive nerve endings were found in close proximity to TNF-producing macrophages especially in the RP. (A) Synaptophysin (green) and TNF (red). (B) Synaptophysin (green) and RP macrophages (blue). (C) Synaptophysin (green), TNF (red), and RP macrophages (blue). (D) Digital magnification of C. (E and F) Sequential slices (10 μm thick) of a normal spleen showing similar spatial distribution of catecholamines (E) and synaptophysin (F). (G and H) Sequential slices of a spleen harvested 7 days after splenic neurectomy stained for catecholamines (G) and synaptophysin (H). (Magnification: A, B, E, F, G, and H, ×100; C, ×630.) Pictures are representative of at least four animals. MZ, marginal zone; WP, white pulp; RP, red pulp.

Fig. 6.

Surgical ablation of the splenic nerve abrogates the TNF-suppressive effect of vagus nerve stimulation. (A) Rats underwent splenic neurectomy (SNVX) or sham-SNVX surgery. Eight to 11 days later, animals received 5 mg/kg LPS i.v. and were subjected to sham surgery or vagus nerve stimulation. Data are presented as mean ± SEM (n = 10–15 per group; *, P < 0.05). (B) Rats received 5 mg/kg reserpine i.p. or vehicle. Twenty-four hours later, rats received 5 mg/kg LPS i.v. and were subjected to vagus nerve stimulation or sham surgery. Data are presented as mean ± SEM (n = 4–6 per group; *, P < 0.05).

Fig. 7.

Anatomical basis of the cholinergic antiinflammatory pathway. Two-neuron model of vagus nerve modulation of cytokine production via the splenic nerve: The preganglionic neuron originates in the dorsal motor nucleus of the vagus; the postganglionic neuron, located in ganglia of the celiac-superior mesenteric plexus, reaches the spleen through the splenic nerve.