Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia

Abstract

TNF has a critical mediator role in inflammation and is an important therapeutic target. We recently discovered that TNF production is regulated by neural signals through the vagus nerve. Activation of this "cholinergic antiinflammatory pathway" inhibits the production of TNF and other cytokines and protects animals from the inflammatory damage caused by endotoxemia and severe sepsis. Here, we describe a role for central muscarinic acetylcholine receptors in the activation of the cholinergic antiinflammatory pathway. Central muscarinic cholinergic activation by muscarine, the M1 receptor agonist McN-A-343, and the M2 receptor antagonist methoctramine inhibited serum TNF levels significantly during endotoxemia. Centrally administered methoctramine stimulated vagus-nerve activity measured by changes in instantaneous heart-rate variability. Blockade of peripheral muscarinic receptors did not abolish antiinflammatory signaling through the vagus nerve, indicating that peripheral muscarinic receptors on immune cells are not required for the cytokine-regulating activities of the cholinergic antiinflammatory pathway. The role of central muscarinic receptors in activating the cholinergic antiinflammatory pathway is of interest for the use of centrally acting muscarinic cholinergic enhancers as antiinflammatory agents.

Fig. 1.

Central administration of muscarinic receptor agonists inhibit systemic TNF in endotoxemic rats. (A) Central administration of muscarine inhibits serum TNF. Rats were injected (i.c.v.) with vehicle (V, saline) or muscarine 1 h before endotoxin (15 mg/kg, i.v.) administration. Serum TNF concentrations were analyzed by ELISA 1.5 h after endotoxin administration (n = 5–7 animals per group). (B) Central administration of the M1 agonist McN-A-343 suppresses serum TNF levels in endotoxemic rats. Animals were injected i.c.v. with vehicle (V) or McN-A-343 1 h before endotoxin (15 mg/kg, i.v.) administration. Serum TNF concentrations were analyzed by ELISA in blood, obtained 1.5 h after endotoxin administration (n = 5–7 animals per group; ∗, P < 0.05).

Fig. 2.

Central treatment with the M2 antagonist methoctramine inhibits serum TNF levels during endotoxemia. Rats were injected (i.c.v.) with vehicle (V, saline) or methoctramine 1 h before endotoxin (15 mg/kg, i.v.) administration. Serum TNF concentrations were analyzed by ELISA 1.5 h after endotoxin administration (n = 5–7 animals per group; ∗, P < 0.05; ∗∗, P < 0.02).

Fig. 3.

Central treatment with methoctramine increases efferent vagus-nerve activity. Animals were treated with vehicle (saline) or methoctramine (500 ng/kg) and ECG recorded for 60 min. High-frequency power (HFP) component of heart-rate variability was determined by spectral analysis (n = 5–7 animals per group; ∗, P < 0.02).

Fig. 4.

Peripheral muscarinic receptors do not mediate the inflammatory response and its regulation by the cholinergic antiinflammatory pathway in endotoxemic rats. (A) Muscarine administered peripherally has no significant effect on serum TNF in endotoxemic rats. Vehicle (V, saline) or sublethal muscarinic doses (50–5,000 ng/kg i.v.) were injected 1 h before endotoxin (15 mg/kg, i.v.) administration. (B) Electrical VNS reduces serum TNF in endotoxemic rats, pretreated with vehicle (V, saline) or AMN (1 mg/kg, i.v.), compared with nonstimulated controls. Rats were pretreated with saline or AMN 30 min before VNS. Serum TNF concentrations were analyzed by ELISA 1.5 h after the administration of a lethal endotoxin dose (15 mg/kg) (n = 5–7 animals per group; ∗, P < 0.01; ∗∗, P < 0.001).

Fig. 5.

Central versus peripheral muscarinic cholinergic regulation of the systemic inflammatory response (proposed mechanism). In rodents, the endotoxin-induced systemic TNF release is inhibited by cholinergic signaling derived from efferent vagus-nerve fibers (4, 5). This cholinergic antiinflammatory pathway (CAP) is mediated through α₇ nicotinic acetylcholine receptors (α₇nAchR) on macrophages (5). Central muscarinic cholinergic activation by stimulation of M1 acetylcholine muscarinic receptors (MAchR, postsynaptic) or inhibition of M2 muscarinic acetylcholine receptors) M2AchR, presynaptic) inhibits systemic TNF release in endotoxemic rats and activates efferent vagus-nerve activity. Activation of the cholinergic antiinflammatory pathway by electrical vagus nerve stimulation reduces systemic TNF levels during endotoxemia through peripheral muscarinic receptor (MAchR)-independent and α₇nAchR-dependent mechanisms (see text for details).