Birkhuser Verlag, Basel, 2003 Inflamm. res. 52 (2003) 291 296 1023-3830/03/070291-06 DOI 10.

1007/s00011-003-1174-8

Inflammation Research

Importance of the vagus nerve for fever and neutrophil migration induced by intraperitoneal LPS injection
M. F. P. Werner 1, D. Fraga 1 , M. C. C. Melo 2, G. E. P. Souza 2 and A. R. Zampronio 1, *
1

Department of Pharmacology, Biological Sciences Section, Federal University of Paran, POBox 19031, 81540-970, Curitiba, PR, Brazil, Fax: ++ 55 41 266 2042, e-mail: azampron@bio.ufpr.br Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, University of So Paulo, 14040-903, Ribeiro Preto, SP, Brazil

Received 21 October 2002; returned for revision 25 November 2002; accepted by I. Ahnfelt-Rnne 19 February 2003

Abstract. Objective: We investigated the importance of the vagus nerve in fever, neutrophil migration and neutrophilia simultaneously induced by intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) and in terms of the production of pre-formed pyrogenic factor (PFPF) and of the fever induced by this factor. Methods: Nave, sham-operated or subdiaphragmatically vagotomized male Wistar rats received either LPS (i.p. or i.pl.) or PFPF (i.v., i.c.v., i.p.). The number of neutrophils was evaluated in peritoneal or pleural fluid and in blood. Fever was monitored using a rectal probe. Results: In nave animals, LPS (0.02200 mg kg1, i.p.) induced dose-related neutrophilia and fever while on neutrophil migration it resulted in a bell-shaped curve. Vagotomy reduced the peritoneal resident cell population (56%), fever (71%) and neutrophil migration (43%) but not the neutrophilia or neutrophil migration to the pleural cavity. Vagotomy did not affect the PFPF production or PFPFinduced fever. Conclusions: Vagus nerve integrity is important not only for fever but also for the neutrophil influx to the peritoneal cavity by controlling the number of resident cells in this cavity. Key words: Vagotomy Fever Neutrophilia Neutrophil migration Introduction The inflammatory response depends on the concerted release of local mediators that are responsible for local vascular and tissue changes as well as for the recruitment of host defence cells [1]. An early key aspect of the acute inflammatory response is the infiltration of polymorphonuclear leukocytes, neutrophils in particular, which depends on the activation/upregulation of adhesive proteins in both neutrophils and endothelial cells in response to the generation of inflammatory cytokines such as interleukin (IL)-1b, tumour necrosis factorCorrespondence to: A. R. Zampronio

a (TNF-a), IL-6, IL-8, platelet-activating factor, leukotriene B4, and complement protein C5a [13]. When formed at sufficiently high rates, some of these mediators reach the blood and can trigger a systemic response named acute phase reaction (APR), which is associated with changes in plasma metal levels, induction of acute-phase proteins, leukocytosis and fever. At least part of the neutrophilia associated with APR reflects the proliferative effects of IL-1b, TNF-a, IL-6 and colony-stimulating factors on granulocyte precursors of the bone marrow [46]. On the other hand, peripherally formed cytokines also convey information to the thermoregulatory centre of the central nervous system (CNS), particularly the hypothalamic pre-optic area, which triggers the febrile response [7]. Studies have suggested that fever depends on cytokine penetration into the brain either through the organum vasculosum laminae terminalis, which lacks the blood brain barrier, and/or their transport via a saturable mechanism [8, 9]. More recently, however, evidence has been obtained suggesting that peripheral afferent nerves, especially those contained in the vagus nerve, play an important signalling role between the inflammatory site and the brain. In this regard, subdiaphragmatic vagotomy blocks fever [10, 11], central induction of IL-1b mRNA [12], hyperalgesia and pain [13] among other responses induced by intraperitoneal (i.p.) injection of either LPS or IL-1b. In view of the above considerations, the aim of the present study was to assess the importance of the vagus nerve for the neutrophil migration and neutrophilia occurring in response to i.p. injection of LPS, in addition to its action on fever. Furthermore, the role of the vagus nerve in the functional activity of resident macrophages through its ability to produce PFPF was also investigated.
Material and methods Animals
Experiments were conducted using male Wistar rats weighing 180 200 g, individually housed at 24 1 C under a 12:12 h light-dark cycle

292 (lights on at 07:00 h) with free access to food chow and tap water until the day of the experiment proper, when only water was made available. All experiments were previously approved by the institutions ethics committee for research on laboratory animals and were performed in accordance with the guidelines for care and animal use set by the National Institutes of Health (USA).

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Inflamm. res.

Experimental design
In the first set of experiments, animals received an intraperitoneal injection of LPS at doses of 0.02 to 200 mg kg1 diluted in sterile saline. Control animals received saline (1 ml/200 g). Blood samples and peritoneal or pleural fluids were collected 6 h later for leukocyte counts as described above. The doses of 0.2 and 200 mg kg1 of LPS were chosen for the remaining experiments. Secondly, sham-operated and vagotomized animals were injected with LPS (0.2 and 200 mg kg1, i. p.) and the febrile response, neutrophilia and neutrophil migration were evaluated. The number of resident cells in the peritoneal cavity of vagotomized animals without any stimulation and the neutrophil migration induced by LPS to the pleural cavity of vagotomized rats were also assessed. Control animals received sterile saline (1 ml kg1). In the next set of experiments, the febrile response was evaluated in nave animals after i.v. (75 mg protein kg1) or i. c. v. (200 ng protein, 3 mL) injection of PFPF produced by macrophages from non-vagotomized and vagotomized animals. PFPF from non-vagotomized animals was also injected intraperitoneally at a dose of 150 mg kg1 in sham-operated and vagotomized animals.

Subdiaphragmatic vagotomy
Animals were anaesthetised with sodium pentobarbital (40 mg kg1, i.p.). The peritoneal cavity was exposed and the ventral and dorsal vagal trunks were removed just below the diaphragm, together with the tissue between the gastric artery and the oesophagus in an extension of 2 cm. Sham-operated animals were submitted to the same procedure without handling the nerves. Animals received antibiotic treatment (oxytetracycline hydrochloride, 400 mg kg1, s.c.) prior to surgery and 2 days after the procedure. To minimise the loss of weight, animals received a complementary liquid caloric meal (13.3% protein, 70.5% carbohydrates, 2.6 % fat, and 4.7% fibers). After the experiments, the stomachs were removed and weighed for an additional assessment of the effectiveness of the surgical procedure. Experiments were conducted 15 days after surgery and animals with abnormal patterns of body weight gain were excluded from the study.

Production of PFPF from LPS-stimulated macrophage monolayers

PFPF was prepared as described by Zampronio et al. [15]. Briefly, rats received an intraperitoneal injection of 10 ml of 3% (p/v) thioglycollate. Peritoneal macrophages were harvested 4 days later using 10 ml of RPMI 1640 medium, pH 7.4, containing 5 U ml1 heparin, and incubated in culture dishes for 1 h at 37C. For the experiments comparing macrophages from vagotomized and non-vagotomized rats, cells were harvested from the peritoneal cavity without previous thioglycollate stimulation. Monolayers of adherent cells (2 106 viable cells per dish) were washed with PBS, stimulated with LPS (10 mg ml1) in RPMI for 30 min at 37C, washed three more times with PBS and incubated with 5 ml of LPS-free medium for 1 h at 37C. The supernatant was concentrated through an Amicon YM30 membrane and the retained portion was resuspended in water without any further purification. After the determination of protein content with a spectrophotometer at 280 nm, the material was lyophilised and stored at 70C until use.

Intracerebral cannula implantation

Under anaesthesia with sodium pentobarbital (40 mg kg1, i.p.), a stainless steel guide cannula (0.7 mm OD, 10 mm long) was stereotaxically placed into the right lateral ventricle [14] and fixed to the skull with jewellers screws embedded in dental acrylic cement. Animals were then promptly treated with oxytetracycline hydrochloride (400 mg kg1, s.c.). All experiments were conducted one week later, after which the animals were anaesthetised (as described earlier) and the location of the cannula track was verified histologically. Animals showing cannula misplacement or blockage upon injection, or abnormal weight gain patterns were excluded from the study.

Temperature measurements Reagents

Body temperature was measured by inserting a thermistor probe (Yellow Springs Instruments no. 402, USA) 3 cm into the rectum, without removing the rats from their home cages, for 1 min at 30 min intervals up to 6 h. The animals were picked up gently and held manually during the temperature measurements. This procedure was performed at least twice on the day before the experiment to minimise changes secondary to handling. On the day of the experiment, the basal temperature of each animal was determined by four measurements at 30 min intervals before any injections. Only animals displaying a mean basal rectal temperature between 36.8 and 37.4C were selected for the study. The experiments were conducted between 08:00 and 17:00 h in a temperature-controlled room (28 1C). The following drugs were employed: Lipopolysaccharide from E. coli (0111:B4), sodium pentobarbital, were from Sigma Chem. & Co., St Louis, USA.; oxytetracycline hydrochloride was from Pfizer, Brazil. Thioglycollate (Difco Lab., Detroit, USA), RPMI (GiBCO Lab., New York, USA), Heparin (Liquemine, Roche, So Paulo, Brazil).

Statistical analysis
For data analysis of change in body temperature, the baseline temperature before any injection was calculated for each animal, and all subsequent temperatures were expressed as change from this average value (DTb). All values are reported as means standard error mean (mean s.e. mean) and were analysed for statistical significance by one-way analysis of variance followed by Bonferronis test. Stomach weight was analysed by Students t test. Significance was set at P < 0.05.

Leukocyte counts
Six hours after i.p. or intrapleural (i. pl.) injection of LPS, animals were anaesthetised with sodium pentobarbitone (40 mg kg1, i.p.) and blood samples were taken by cardiac puncture for leukocyte counts using heparinized syringes. Immediately after blood collection, the animals were killed by cervical dislocation and the peritoneal or pleural cavity was washed with 10 or 5 ml of phosphate-buffered saline (PBS), respectively, containing 0.03% (p/v) bovine albumin and 5 U ml1 heparin. Total and differential cell counts were performed under light microscopy and the results are reported as the number of cells per ml of blood or peritoneal/pleural fluid.

Results Six hours after intraperitoneal injection of sterile saline in nave animals no significant variation in cell numbers was observed in the peritoneal cavity or in blood. In contrast, LPS at the dose range of 0.02 to 200 mg kg1 promoted a signifi-

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Fig. 1. Neutrophil migration to the peritoneal cavity and blood neutrophilia induced by LPS. Animals received sterile saline (Sal) or LPS i. p. at the doses indicated in the figures. The number of neutrophils was evaluated in the peritoneal fluid (panel A) and in the blood (panel B) 6 h after injection. Bars show the mean s.e.mean of the number of neutrophils.106 ml-1 in 7 11 animals. * p < 0.05 compared to the saline group; # p < 0.05 compared to the LPS 0.02 and 0.2 groups.

cant bell-shaped neutrophil migration (Fig. 1A). Since there was no statistically significant difference between doses, the dose that produced the maximal neutrophil migration, of 0.2 mg kg1, was selected for the remaining experiments. The neutrophil migration to the peritoneal cavity was accompanied by an increase in the number of circulating neutrophils (neutrophilia, 6 h) though in a different pattern. The neutrophilia increased dose-dependently up to the highest dose used (Fig. 1B). The highest dose (200 mg kg1), which allows us to observe a full response of blood neutrophilia, was then selected. The doses selected for neutrophil migration and blood neutrophilia were assessed for their ability to cause a febrile response. The febrile response induced by 200 mg kg1 LPS started 2 h after injection, peaked at 2.5 h and lasted until to the end of the experimental period, while the dose of 0.2 mg kg1 promoted a very low fever that lasted about 1 h. Vagotomized and sham-operated animals did not show significant temperature changes after saline injection. Nonetheless, vagotomized animals that received LPS (0.2 or 200 mg kg1 i.p.) did not develop fever, contrasting with the respective sham-operated group (Fig. 2). The effectiveness of subdiaphragmatic vagotomy was also evaluated by the removal and weighing of the stomach. Vagotomized animals showed a significantly higher stomach weight (2.07 0.72 g) than sham-operated animals (1.36 0.23 g). Subdiaphragmatic vagotomy did not affect the viability of resident cells in the peritoneal cavity evaluated by the dye exclusion test (88% viable cells in sham-operated animals and 84.3% in vagotomized animals). However, the number of resident mononuclear cells found in the peritoneal cavity of vagotomized animals was 56% lower than that found in sham-operated animals (Fig. 3, left panel). Surgical handling was not the cause of mononuclear reduction in vagotomized animals since in sham-operated animals the number of these cells was not different from nave animals (data not shown).

Fig. 2. Subdiaphragmatic vagotomy abolishes the febrile response induced by LPS. LPS, 0.2 mg kg1 (panel A) or 200 mg kg1 (panel B), was injected i.p. in vagotomized (VGX) and sham-operated animals (SHAM). Control animals received the same volume of sterile saline (Sal). Values show the mean s.e.mean of the change in rectal temperature observed in 513 rats. * p < 0.05 compared to the SHAM/Sal group, #p < 0.05 compared to the SHAM/LPS group.

Fig. 3. Subdiaphragmatic vagotomy reduces the number of peritoneal resident mononuclear cells and the neutrophil migration induced by LPS. The left panel shows the number of resident cells in the peritoneal cavity of sham-operated (SHAM) and vagotomized (VGX) rats. The right panel shows the number of neutrophils in the peritoneal cavity 6 h after sterile saline (Sal) or LPS, 0.2 mg kg1, in VGX and sham-operated animals (SHAM). Bars show the mean s.e.mean of the number of cells 106 ml1 washing fluid in 511 animals. *p < 0.001 compared to the SHAM group or to the respective saline groups; #p < 0.01 compared to the SHAM-LPS group.

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Fig. 4. Subdiaphragmatic vagotomy does not modify neutrophilia induced by LPS. Vagotomized (VGX) and sham-operated animals (SHAM) received an i.p. injection of sterile saline (Sal) or LPS, 200 mg kg1, and the blood was collected by cardiac puncture after 6 h. Bars show the mean s.e.mean of the number of neutrophils.106 ml1 in 12 15 animals. *p < 0.05 compared to the respective saline groups.

Fig. 6. PFPF induces fever in vagotomized animals (VGX). PFPF from macrophages of non-vagotomized rats (PFPF, 50 mg kg1, i. p.) was injected in VGX and sham-operated animals (SHAM). Control animals received the same volume of sterile saline (Sal). Values show the mean s.e.mean change in rectal temperature observed in 513 rats. * p < 0.05 compared to the respective saline group.

trophils 106 ml1 against 7.14 0.83 neutrophils 106 ml1, respectively). Unlike neutrophil migration, the number of circulating neutrophils 6 h after i.p. injection of 200 mg kg1 LPS did not differ between vagotomized and sham-operated animals (Fig. 4). PFPF obtained from macrophages of vagotomized and non-vagotomized animals injected i.v. or i.c.v. induced a similar febrile response when the same number of macrophages was placed on the plates (Fig. 5). Also, PFPF obtained from macrophages of non-vagotomized animals induced a significant febrile response when injected i.p. in vagotomized or sham-operated animals (Fig. 6). Discussion In the last few years some studies have shown that the information generated by peripheral damaging stimuli might reach the central nervous system via peripheral sensory nerves, including certain fibres in the vagus nerves [for a review see 16]. Our results confirm previous studies showing that subdiaphragmatic vagotomy blocks the development of fever [10, 11, 17]. Nevertheless, we showed here for the first time a reduction in the number of resident cells in the peritoneal cavity of subdiaphragmatically vagotomized rats. Resident cells in peritoneal cavities mainly consist of macrophages [18] and it is plausible to hypothesise that the efferent vagus nerve modulates the release of mature macrophages from milky spots localized in the omentum, since this tissue is largely innervated by the vagal hepatic branch [19]. In vitro and in vivo studies have demonstrated that mouse milky spots have a microenvironment in which precursor cells of the mononuclear phagocyte system can home and proliferate, illustrating that milky spots play a role as a source of local macrophage generation, e.g. that of free peritoneal macrophages [20]. Other studies have also shown that milky spots seem to be essential for the maturation (development and differentiation) of omental macrophages [21, 22]. Supporting this hypothesis is the evidence that efferent vagus nerve signalling may facilitate lymphocyte release from the thymus through a nicotinic acetylcholine (ACh) receptor [23].

Fig. 5. Macrophages from vagotomized (VGX) rats stimulated with LPS produce PFPF. PFPF (75 mg kg1, i. v., panel A or 200 ng, i.c.v., panel B) from macrophages of VGX rats (VGX-PFPF) or from nonvagotomized animals (N-PFPF) were injected in the animals. Control rats received the same volume of the respective vehicle (saline, Sal or CSF). Values show the mean s.e.mean of the change in the rectal temperature observed in 5 13 rats. *p < 0.05 compared to the Saline and CSF-treated group, p < 0.05.

Neutrophil migration to the peritoneal cavity 6 h after 0.2 mg kg1 LPS injection was also significantly reduced (43%) in vagotomized animals compared to sham-operated animals (Fig. 3, right panel). Contrasting with the cellular migration to the peritoneal cavity, the number of neutrophils that migrated to the pleural cavity of subdiaphragmatically vagotomized animals 6 h after 0.2 mg kg1 of LPS administered into the pleural cavity was not significantly different from that observed in sham-operated animals (8.59 1.7 neu-

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In addition to reducing the number of resident cells in the peritoneal cavity, subdiaphragmatic vagotomy also considerably reduced the neutrophil migration to this cavity in response to LPS. The selectivity of subdiaphragmatic vagotomy and the importance of local vagal innervation for neutrophil migration were evidenced here by the demonstration that neutrophil migration to a site other than the peritoneal cavity, e.g. the pleural cavity, was unchanged. This is also true for fever installation since intramuscular injection of LPS results in identical fever in both vagotomized and shamoperated guinea pigs [11]. We showed before that resident peritoneal macrophages act as alarm cells by controlling neutrophil migration in response to several inflammatory stimuli since there is a direct relationship between the number of peritoneal cells and the neutrophil migration to the inflamed peritoneal cavity [24, 25]. On this basis, it seems reasonable to suggest that vagal nerves, by controlling the peritoneal mononuclear cell population, directly control the neutrophil migration to the peritoneal cavity, which may constitute an additional protective pathway against localized invasion of damaging agents. It has been shown that neuropeptides, such as substance P (SP), calcitonin gene-related peptide and somatostatin, may modulate macrophage activity [2628]. For instance, SP can induce the expression of Bcl-2 and NO which may contribute to prevent the signals of apoptosis in macrophages. Considering that 75% of the nerve fibres in subdiaphragmatic trunks are afferent sensitive fibers which can release neuropetides [for a review see 29], we cannot discard their involvement in the development and maintenance of resident cell population in the peritoneal cavity. This possibility needs further investigation but could also explain the reduced number of these cells in the peritoneal cavity of vagotomized rats. Our data contrast somewhat with that from Borovikova et al. [30] which described an in vivo anti-inflammatory pathway in a model of septic shock induced by a high (15 mg kg1) dose of LPS intravenously injected in rats. In the cited study, cervical vagotomy increased serum TNF levels and hepatic TNF production during lethal endotoxemia while electrical stimulation of the distal vagus nerve decreased LPS-stimulated TNF synthesis in the liver and attenuated the hypotension. Recently, the same group [31], confirmed that a nicotinic ACh receptor regulates the cytokine synthesis. These authors showed that electrical stimulation of the vagus nerve inhibits TNF release in wild-type mice, but fails to inhibit TNF synthesis in nicotinic receptor a7 subunit-deficient mice. In addition, Romanovsky [32] described a hepatoceliac vagal anti-inflammatory pathway, presumably efferent, which counteracts the development of LPS-induced hypothermia and shock. Unlike neutrophil migration, the number of circulating neutrophils in vagotomized and sham-operated rats after LPS was the same, suggesting that subdiaphragmatic vagotomy did not affect the level of circulating cytokines such as IL-1 and IL-6, which are known to cause granulocytopoiesis by their myeloproliferative and differentiating effects in the bone marrow [4, 33, 34]. Moreover, our data are supported by the findings of Romanovsky et al. [35] and Hansen et al. [36] who showed that, at lower doses of LPS than that used by Borovikova et al. [30], subdiaphragmatic vagotomy itself has no effect on the circulating levels of IL-1 and IL-6. This fact

suggests that, at lower doses of LPS, an anti-inflammatory cholinergic pathway was not activated. We also evaluated the ability of macrophages from vagotomized animals to release PFPF, a proteic pre-formed pyrogenic factor released from LPS-stimulated cells, even after treatment with dexamethasone or cycloheximide [37]. Macrophages from vagotomized animals still can release PFPF in a sufficient amount to induce fever when injected i.v. or i.c.v. in nave animals as long as we equalise the number of macrophages to produce the protein. Thus, the reduction in the number of resident cells could reduce the production of pre-formed mediators such as PFPF, contributing to the lack of fever in vagotomized animals. Also, PFPF produced by macrophages from non-VGX animals induced fever even when injected i.p. in VGX animals. Conversely, subdiaphragmatic vagotomy blocked fever induced by intraperitoneally injected IL-1b [10]. We cannot exclude the possibility that this difference between IL-1b and PFPF may be related to the dose since at this dose it is possible that PFPF gains the systemic circulation and reaches the hypothalamus. An interesting aspect of this result is that it confirms that effector mechanisms for fever development were not affected by vagotomy [11, for a review see 16]. In conclusion, our data confirm the importance of vagus nerve in the febrile response and, demonstrate by the first time, that vagus nerve contributes to bring about the neutrophil migration induced by LPS to the peritoneal cavity by controlling the resident cell population in this cavity and therefore the production of mediators that promote neutrophil influx and fever. Finally, this study extends previous evidence of the integrative pathway between the defense cells in the periphery and the CNS.
Acknowledgements: We thank the Fundao de Amparo Pesquisa do Estado de So Paulo for financial support (FAPESP, Proc. Nr. 97/09837-6). M.F.P.W. and D.F. were recipients of fellowships from Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq, PIBIC, 19982000).

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