Via β-adrenoceptors, stimulation of extrasplenic sympathetic nerve fibers inhibits lipopolysaccharide-induced TNF secretion in perfused rat spleen

https://doi.org/10.1016/j.jneuroim.2003.09.011Get rights and content

Abstract

Using a spleen slice microsuperfusion technique in mice, we have previously characterized the role of norepinephrine (NE) and other neurotransmitters for sympathetic modulation of IL-6 and TNF secretion of splenic macrophages. Since experiments in spleen slices do not reflect the situation of an entire perfused organ, we investigated sympathetic modulation of lipopolysaccharide (LPS)-induced secretion of IL-6 and TNF in perfusion experiments of rat spleen. In an organ bath, perfusion was performed in explanted whole spleens, and catecholamines and cytokines were measured by HPLC and ELISA, respectively. Release of NE depended on stimulation frequency (maximum at 10 Hz). Apart from NE, perfusates also contained significant amounts of dopamine and epinephrine. Furthermore, perfusate epinephrine levels correlated positively with perfusate NE levels (RRank=0.750, p<0.001) but not with plasma epinephrine concentrations. This indicates that epinephrine is a conversion product of sympathetically released NE. Early electrical stimulation of extrasplenic splenic nerves, 20 min after administration of LPS, significantly inhibited TNF secretion. This electrically induced effect was abrogated by simultaneous administration of propranolol (10-6 M) but it was not influenced by administration of either an α1- or α2-adrenergic antagonist. Late electrical stimulation of splenic nerves, 2.5 h after administration of LPS, did not change TNF secretion. Interestingly, no influence of early or late sympathetic nerve fiber stimulation on IL-6 secretion was observed. In conclusion, this is the first perfusion study of the entire spleen that demonstrates that early electrical stimulation of sympathetic splenic nerve fibers directly inhibits LPS-induced TNF secretion. This study corroborates the idea that splenic sympathetic nerves are able to inhibit important activators of the innate immune system when stimulation happens very early or even prior to their induction by LPS.

Introduction

Since the first immunohistochemical studies of sympathetic nerves in the vicinity of immune cells in the spleen (for review, Bellinger et al., 2001), the functional interplay of these nerves and immune cells in the spleen has been described in several reports Straub et al., 1998b, Elenkov et al., 2000, Madden, 2001. Most often norepinephrine (NE) has been in the focus of experiments in order to study the functional interaction between sympathetic nerves and local immune cells. We previously described the modulation of splenic IL-6 secretion by locally released NE, opioids, neuropeptide Y (NPY), ATP, and adenosine using a spleen slice microsuperfusion technique Straub et al., 1997, Straub et al., 2000b, Straub et al., 2002. With respect to NPY and adenosine, features of co-transmission with NE for regulation of IL-6 secretion were observed Straub et al., 2000b, Straub et al., 2002. Furthermore, IL-6 secreting cells in these spleen slices are most probably macrophages because treatment of mice with clodronate liposomes, which destroys splenic macrophages but no other splenic cells, abolished IL-6 secretion from spleen slices (Straub et al., 1998a). In further experiments, the role of sympathetic modulation of splenic TNF secretion was scrutinized by using a highly potent monoclonal anti-TNF antibody and TNF receptor knock out mice (Straub et al., 2000a). From these studies, a complex picture of neuroimmunomodulation appears according to which regulation of IL-6 secretion depends on prior modulation of TNF secretion (Straub et al., 2000a).

Since experiments with spleen slices do not completely reflect investigations of an entire organ, our earlier results possibly cannot be compared to the outcome of perfusion experiments due to important factors such as (A) direct influence of the electrical current on macrophages, (B) uncontrolled medium flow through splenic tissue (in comparison to vessels), (C) another type of exchange between the interstitial and intravascular space, (D) missing modulation of endothelial cells by shear stress, (E) lack of vascular constriction and dilation, and many others. In order to get a more integral picture of sympathetic modulation of splenic cytokine secretion, perfusion experiments were carried out. At an early and late time point in relation to administration of lipopolysaccharide (LPS), splenic sympathetic nerve fibers were electrically stimulated far away from immunocompetent cells outside the spleen. These studies should add to the knowledge of neuroimmunomodulation of early cytokine secretion in the spleen.

Section snippets

Animals

Male CD-Rats were purchased from Charles River (Sulzfeld, Germany) and housed at 22 °C and 70% air humidity on a 12-h light–dark cycle with free access to water and standard food. At the time of experiment, they weighed approximately 200 g. Experiments were performed according to the Guidelines for Animal Experimentation (Government of the Oberpfalz, AZ 621-2531.1-19/00).

Perfusion of the spleen

Animals were anesthetized using 12 mg/kg ketamine and 1 mg/kg xylazine. The spleen was prepared according to a previously

Catecholamine release from perfused rat spleen

Norepinephrine was released from the isolated perfused spleen in a frequency-dependent manner (Fig. 2A), and maximum release was observed at 10 Hz. Thus, in all further experiments stimulation was performed with 10 Hz. Norepinephrine secretion was accompanied by simultaneous release of epinephrine and dopamine (Fig. 2B). The time-concentration curve of norepinephrine and epinephrine were very similar (Fig. 2B), whereas the maximum of dopamine release appeared 1 h later (Fig. 2B). Concentration

Discussion

These studies in perfused rat spleen were carried out in order to investigate the early and late influence of electrical stimulation of splenic sympathetic nerves on spontaneous and LPS-induced secretion of IL-6 and TNF. These two cytokines were selected because we wanted to compare our earlier experimental conditions in mice spleen slices, which focused on IL-6 and TNF, with extrasplenic stimulation of sympathetic nerves under perfusion conditions Straub et al., 1997, Straub et al., 1998a,

Acknowledgments

This study was supported by the Deutsche Forschungsgemeinschaft (Str 511/9-1). We thank Stefan Grüne for his technical advice concerning perfusion experiments.

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