Cholinergic neural signals to the spleen down-regulate leukocyte trafficking via CD11b

Abstract

The cholinergic anti-inflammatory pathway is a physiological mechanism that inhibits cytokine production and diminishes tissue injury during inflammation. Recent studies demonstrate that cholinergic signaling reduces adhesion molecule expression and chemokine production by endothelial cells and suppresses leukocyte migration during inflammation. It is unclear how vagus nerve stimulation regulates leukocyte trafficking because the vagus nerve does not innervate endothelial cells. Using mouse models of leukocyte trafficking, we show that the spleen, which is a major point of control for cholinergic modulation of cytokine production, is essential for vagus nerve-mediated regulation of neutrophil activation and migration. Administration of nicotine, a pharmacologic agonist of the cholinergic anti-inflammatory pathway, significantly reduces levels of CD11b, a beta(2)-integrin involved in cell adhesion and leukocyte chemotaxis, on the surface of neutrophils in a dose-dependent manner and this function requires the spleen. Similarly, vagus nerve stimulation significantly attenuates neutrophil surface CD11b levels only in the presence of an intact and innervated spleen. Further mechanistic studies reveal that nicotine suppresses F-actin polymerization, the rate-limiting step for CD11b surface expression. These studies demonstrate that modulation of leukocyte trafficking via cholinergic signaling to the spleen is a specific, centralized neural pathway positioned to suppress the excessive accumulation of neutrophils at inflammatory sites. Activating this mechanism may have important therapeutic potential for preventing tissue injury during inflammation.

FIGURE 1

Administration of cholinergic agonist inhibits leukocyte trafficking in the presence of a spleen. Mice underwent sham surgery (A and C) or splenectomy surgery (B and D) before the carrageenan air pouch model (A and B) or CLP-induced sepsis (C and D) to assess leukocyte trafficking (as described in the Materials and Methods section). A and B, Mice receive either saline (solid) or nicotine (1 mg/kg, i.p., hatched) 15 min before carrageenan challenge (in a dorsal air pouch). Five hours later, mice were euthanized and the leukocytes in the air pouch were collected and enumerated. C and D, Mice received saline (solid) or nicotine (0.4 mg/kg, i.p., hatched) 15 min, 6 h, and 18 h post CLP. After 24 h, mice were euthanized and the leukocytes present in the peritoneal cavity were collected and enumerated. Data from three experiments (five to six animals per group) are shown as inflammatory cells (percentage of control) (mean ± SEM), with 100% = sham-vehicle or splenectomy-vehicle. **, p < 0.01 comparing treatment to vehicle control.

FIGURE 2

Cholinergic agonist treatment decreases the appearance of CD11b on the surface of neutrophils. A, Mice received either saline or nicotine (0.1–1 mg/kg, i.p.) and blood was collected 1 h later. CD11b expression by circulating neutrophils was determined by flow cytometry as described in the Materials and Methods section. Data shown are neutrophil surface CD11b levels (mean fluorescence intensity, MFI) ± SEM. B and C, Mice received either saline (solid) or nicotine (1 mg/kg, i.p., hatched) 1 h before collection of whole blood and ex vivo stimulation with either LPS (B) or fMLP (C) for 15 min at 37°C as described in the Materials and Methods section. Data from three experiments (five to six mice per group) were combined and data are shown as neutrophil surface CD11b levels (mean fluorescence intensity, MFI) ± SEM. **, p < 0.01 comparing treatment to appropriate control.

FIGURE 3

Cholinergic stimulation inhibits neutrophil surface CD11b levels in the presence of a spleen. Mice underwent sham (black) or splenectomy (SPLX, gray) surgery 1 wk before administration of nicotine (1 mg/kg, i.p). One hour later, blood was collected to determine the levels of CD11b on surface of neutrophils. Data from three experiments (three to five mice per group) were combined and data are shown as percentage of change in neutrophil surface CD11b levels in sham vs splenectomized mice (induced by nicotine treatment) ± SEM. *, p < 0.05 comparing the percentage of change in neutrophil CD11b levels in sham vs splenectomized mice.

FIGURE 4

VNS-mediated suppression of leukocyte recruitment and neutrophil surface CD11b levels is dependent on the spleen. Mice underwent sham surgery (A) or splenectomy (SPLX) (B) surgery 1 wk before sham surgery (solid) or vagus nerve stimulation (VNS, hatched); 15 min later, carrageenan was injected into dorsal air pouches. Leukocyte accumulation within air pouches was determined 5 h later. Data are expressed as inflammatory cells, percentage control (mean ± SEM) where 100% is either sham control or splenectomy/sham control (5 mice per group). C and D, Mice underwent either sham surgery (solid) or vagus nerve stimulation (VNS, hatched) 1 h before analysis of CD11b expression on circulating neutrophils following ex vivo LPS (C) or fMLP (D) stimulation. Data from three experiments (three to five mice per group) were combined and data are shown as neutrophil surface CD11b levels (mean fluorescence intensity, MFI) ± SEM. *, p < 0.05; **, p < 0.01 comparing treatment to appropriate control.

FIGURE 5

VNS-mediated inhibition of LPS-mediated neutrophil surface CD11b levels requires splenic innervation. Mice underwent sham surgery (black) or splenic neurectomy (SNVX) (gray) 7–12 days before sham surgery (solid) or vagus nerve stimulation (VNS, hatched). One hour later, peripheral blood was collected and either left untreated or stimulated with LPS ex vivo. Data from two experiments (three to five mice per group) were combined and data are shown as CD11b surface levels (fold-increase following LPS stimulation, based on flow cytometry data) (mean ± SD) (A). *, p < 0.05; **, p < 0.01 comparing sham-sham vs sham-VNS and CD11b surface levels (on peripheral blood neutrophils) (B) determined by flow cytometry methods comparing basal (solid line) vs LPS-stimulated (hatched line), with isotype control shown as a shaded histogram.

FIGURE 6

Cholinergic agonist regulates neutrophil surface CD11b levels through suppressing F-actin polymerization. Mice received saline (solid) or nicotine (1 mg/kg, i.p., hatched) 1 h before analysis of F-actin polymerization by isolated blood neutrophils determined by FITC-phalloidin binding and flow cytometry methods. Data from three experiments are shown as the relative F-actin content induced following incubation with 0.1 μM fMLP (mean ± SEM). **, p < 0.01 comparing treatment to vehicle.

FIGURE 7

Cholinergic stimulation increases neutrophil localization to the spleen during acute inflammation. Splenic (A) neutrophils and T lymphocytes (B) were enumerated 0, 15, and 30 min following vehicle (saline, solid line) or nicotine treatment (1 mg/kg, i.p., hatched line). Data from two experiments are expressed as total number of specific cells per spleen (mean ± SD). *, p < 0.05, comparing treatment levels to baseline levels.

FIGURE 8

Proposed model for the role of the spleen in regulating leukocyte trafficking via cholinergic signaling. Collectively, our findings reveal a centralized pathway for controlling leukocyte trafficking to sites of inflammation via modulation of neutrophil CD11b levels through a spleen-dependent neural cholinergic signaling pathway. This pathway can be manipulated by cholinergic stimulation via electrical stimulation of the vagus nerve or by pharmacological cholinergic agonists to attenuate excess leukocyte accumulation at the site of inflammation and reduce tissue damage.