Leukotriene synthesis inhibitor decreases vasopressin release in the early phase of sepsis

Journal of Neuroimmunology 238 (2011) 52–57

Contents lists available at ScienceDirect

Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Leukotriene synthesis inhibitor decreases vasopressin release in the early phase of sepsis Thalita Freitas Martins a, Carlos Artério Sorgi b, Lúcia Helena Faccioli b, Maria José Alves Rocha a,⁎ a Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto; Universidade de São Paulo, Avenida do Café s/n, CEP 14040-900, Ribeirão Preto, SP, Brazil b Departamento de Análises Clínicas, Toxicológicas e Bromatológicas da Faculdade de Ciências Farmacêuticas de Ribeirão Preto; Universidade de São Paulo, Avenida do Café s/n, CEP 14040-900, Ribeirão Preto, SP, Brazil

a r t i c l e

i n f o

Article history: Received 17 May 2011 Received in revised form 25 July 2011 Accepted 1 August 2011 Keywords: Infection Inflammation Nitric oxide Hypotension Vasopressor Hormone

a b s t r a c t The aim was to analyze the effect of leukotriene synthesis inhibitor administered intraperitoneally in vasopressin release during sepsis. Male Wistar rats received injections of MK-886 (1.0, 2.0 or 4.0 mg/kg) or vehicle (DMSO 5%) 1 h before cecal ligation and puncture. There was some variation on the survival rate depending on the dose used but the drug did not modify the hematocrit, osmolality, serum sodium and nitrate, plasma protein, and neutrophil recruitment, in any dose. Nevertheless, vasopressin (AVP) release decreased in a dose–response manner in the early phase of sepsis. These results support the suggestion that leukotrienes (LTs) are involved in AVP release during sepsis. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Clinical and experimental studies report that in the initial phase of sepsis, high AVP levels can be found in plasma of patients or animals. This can be interpreted as a systemic attempt, to restore blood pressure that tends to decrease due to cytokines and NO release. Notwithstanding, in the late phase, when the observed hypotension would be expected to stimulate AVP secretion, its plasma levels were inappropriately low (Landry et al., 1997; Landry and Oliver, 2001; Sharshar et al., 2003). Low plasma AVP levels can be considered a deleterious consequence of an abnormal pituitary response due to baroreflex impairment and high levels of nitric oxide that will also contribute to the hypotension, progression of sepsis, multiple organ failure and death (Landry et al., 1997; Holmes et al., 2001; Sharshar et al., 2003; Maxime et al., 2007). Understanding in detail the mechanisms responsible for the release of this hormone during sepsis should, thus, be of clinical importance. During sepsis, proinflammatory cytokines are released in an uncontrolled manner, and a large number of these, in particular tumor necrosis factor (TNF)-α, interleukin (IL)-1α and interferon (IFN)-γ, as well as nitric oxide (NO), have been implicated in the pathophysiological process of this disease (Rios-Santos et al., 2003;

⁎ Corresponding author. Tel.: + 55 16 3602 3974; fax: + 55 16 3633 0999. E-mail address: [email protected] (M.J.A. Rocha). 0165-5728/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2011.08.001

Benjamim et al., 2005; Sriskandan and Altmann, 2008). At present, a growing body of evidence suggests that also lipid-derived inflammatory mediators, such as leukotrienes (LTs), may play a key role in sepsis-associated disorders (Sprague et al., 1989; Morlion et al., 2000; Benjamim et al., 2005; Baenkler et al., 2006). The capacity for complete biosynthesis of LTs from arachidonic acid (AA) is largely confined to leukocytes, and the biosynthesis pathway can be triggered by a variety of stimuli, including lipopolysaccharide (LPS), IL-1 and TNF-α. These stimuli activate signal transduction cascades that, in turn, activate LTs-forming enzymes (Clark et al., 1995; Hirabayashi and Shimizu, 2000). Briefly, a cytosolic phospholipase A2 (cPLA2) initiates the synthesis of LTs by cleavage of AA from membrane phospholipids. The subsequent interaction of AA with 5-lipoxygenase (5-LO) and 5-lipoxygenase activating protein (FLAP) forms the intermediate 5-HPETE (5-hydroxyperoxy-6,8,11,14-eicosatetraenoic acid), which is rapidly converted to LTA4. This unstable intermediate can be hydrolyzed to form LTB4 or it can be conjugated with glutathione by LTC4 synthase to produce the cysteinyl leukotrienes (cys-LTs) LTC4, LTD4 and LTE4 (Peters-Golden and Brock, 2003; Peters-Golden and Henderson, 2007). Furthermore, LTA4 generated in one cell can be taken up by another one that expresses LTA4 hydrolase or LTC4 synthase to there continue its biochemical transformation (Murphy and Gijon, 2007). This process named transcellular biosynthesis suggests that the cellular environment exerts an important control over LTs production (Di Gennaro et al., 2004). In functional terms, LTB4 promotes chemotaxis, activation and adhesion of leukocytes to endothelial cells, thus being the

T.F. Martins et al. / Journal of Neuroimmunology 238 (2011) 52–57

mediator responsible for recruiting leucocytes from the circulation to the infection locus (Cunningham et al., 1980; Ford-Hutchinson et al., 1980; Gimbrone et al., 1984; Flamand et al., 2007). In contrast, the cys-LTs are better known for their bronchoconstriction effects, characteristic symptoms of patients with asthma (Dahlen et al., 1980), and in blood vessels they increase vascular permeability and cause hypotension (Dahlen et al., 1980; Smedegard et al., 1982; Henderson and Klebanoff, 1983; Goulet et al., 2000; Flamand et al., 2007). Cys-LTs are also involved in a complex network of interactions with a variety of inflammatory mediators, such as NO. Several studies have demonstrated the effect of inhibitors or antagonists of LTs in NO production. Montelukast (antagonist of CysLT1 receptor) has been shown to reduce levels of exhaled NO in clinical trials with asthmatic adults (Wilson et al., 2001; Sandrini et al., 2003) and children (Bisgaard et al., 1999; Bratton et al., 1999). This antagonist was also shown to reduce the expression of iNOS in the lung of rats (Knigge et al., 2003). In glial cells, the MK-886 (an LT biosynthesis inhibitor) was responsible for reducing the LPS-induced iNOS expression and NO production, suggesting that 5-LO mediates iNOS gene expression during endotoxemia (Won et al., 2005). A growing body of evidence suggests that LTs also play an important role in the central nervous system (Lindgren et al., 1985; Shimada et al., 2005) due to the expression of 5-LO and FLAP protein (Lammers et al., 1996), as well as the expression of CysLT2 (cys-LT receptor) in several brain regions, including the hypothalamus (Heise et al., 2000). One of the first reports describing a biological effect of LTs in the brain involved the neuroendocrine system. In vitro studies showed that the release of luteinizing hormone (LH) from pituitary cells in response to gonadotrophins releasing hormone (GnRH) was partly mediated by LTs. The response appears to be LTC4 specific since LTB4 did not alter the release of LH (Hulting et al., 1985). In the brain, LTC4 also seems to be related with the vasopressinergic system of the hypothalamic–neurohypophyseal axis. LTC4 synthase, but not LTA4 hydrolase, was selectively localized in vasopressinergic neurons of the hypothalamic paraventricular, supraoptic and suprachiasmatic nuclei and in the retrochiasmatic area. In addition it was detected in axons emanating from these neurons to the neurohypophysis (Shimada et al., 2005). In a recent study from our laboratory we suggested that LTs are involved in arginine vasopressin (AVP) release. The central administration of an inhibitor of leukotriene synthesis in rat abolished the secretion of AVP in the initial phase of the sepsis. Furthermore, its injection improved survival and attenuated the increase of LTC4 enzyme in the hypothalamus and of serum nitrate levels seen during sepsis (Athayde et al., 2009). As already shown, NOS inhibitor may also affect vasopressin secretion during sepsis, though the results depend on the route of inhibitor administration (Carnio et al., 2006; Corrêa et al., 2007). The objective of this work was to investigate the effects of an intraperitonially administered inhibitor of leukotriene synthesis on AVP release and NO production in the course of experimental sepsis.

53

in high suffering immediately before or soon after the time-points defined in this study. 2.2. Experimental protocol The animals received an intraperitoneal (i.p.) injection of 1 ml containing dimethyl sulfoxide (DMSO) 5% as vehicle or an inhibitor of leukotriene synthesis, MK-886 (3-[1-(p-chlorobenzyl)-5-(isopropyl)3-tert-butylthioindol-2-yl]-2,2-dimethylpropanoic acid, Merck Frosst Canada Ltd., Quebec, Canada) 1 h before CLP or sham operation. The biological half life of MK-886 is reported to be 2 h (Friedman et al., 1993). In the first group of animals (n = 80), the survival of the animals was monitored for 3 days. In the second group (n = 200) the animals were decapitated at 0, 4 (early phase of sepsis) or 24 h (late phase of sepsis) after surgery and blood was collected for determination of hematocrit, serum nitrate and sodium, plasma osmolality and proteins, and plasma AVP measurements. The peritoneal exudate was carefully collected for evaluating neutrophil migration into the peritoneal cavity. 2.3. Cecal ligation and puncture (CLP) Severe sepsis was induced by CLP, as previously described (Corrêa et al., 2007; Pancoto et al., 2008; Athayde et al., 2009; Oliveira-Pelegrin et al., 2009). Briefly, rats were anesthetized with 2, 2, 2-tribromoethanol (Acros Organics, Geel, Belgium, 250 mg/kg i.p.). Under sterile surgical conditions, a 2 cm midline incision was made on the ventral surface of the abdomen, and the cecum was exposed and ligated below the ileocecal junction without causing bowel obstruction. The cecum was punctured 10 times with a 16-gage needle, and fecal contents were allowed to spill into the peritoneum. The cecum was repositioned in the abdomen, and the peritoneal wall and skin incisions were closed. Sham animals underwent an identical laparotomy, but did not undergo ligation and puncture and served as controls. All animals received a subcutaneous injection of saline (20 ml/kg body weight) immediately after the surgery. The animals were allowed to recover in their cages with free access to food and water. 2.4. Neutrophil migration into the peritoneal cavity Neutrophil migration was assessed 4 h after CLP. The animals were killed and the cells present in the peritoneal cavity were harvested by introducing 15 ml of phosphate-buffered saline (PBS). Total counts were performed with Turk solution in a Neubauer chamber. Differential cell counts were obtained using a cytocentrifuge (Cytospin 3, Shandon Southern Products Ltd., Cheshire, UK) and staining with Panotico (Laborclin LTDA, Pinhais Parana, Brazil). The results were expressed as the number of neutrophils per ml of peritoneal exudate. 2.5. Determination of hematocrit, serum nitrate and sodium, plasma osmolality and protein

2. Material and methods 2.1. Animals Male Wistar rats (250 ± 30 g) from the Animal Care Facility of the Universidade de São Paulo (USP), Campus Ribeirão Preto were used in the present study. The rats were housed in controlled temperature (25 ± 1 °C) and photoperiod (12-h light/dark cycle) conditions, with food (Nuvilab CR-1, Nuvital Nutrientes, Paraná, Brazil) and water available ad libitum. All experimental protocols were performed according to the international guidelines on the ethical use of animals and approved by the Animal Ethics Committee of USP (CEUA)Campus Ribeirão Preto, defining the number of animals used and minimizing their suffering. Humane endpoints in shock research (Nemzek et al., 2004) were used as criteria to euthanize CLP animals

Hematocrit was measured by centrifugation, serum sodium by flame photometry (Micronal, São Paulo, Brazil), and plasma osmolality by freezing-point depression (Precision System, Inc., Natick, MA, USA). Plasma protein was determined by a Bradford colorimetric assay in a Microplate reader (Bio-Rad Laboratories, Hercules, CA, USA), and nitrate was quantified by chemoluminescence system (Sievers 280 NOA, Sievers, Boulder, CO, USA). 2.6. Radioimmunoassay (RIA) for AVP A RIA for AVP was performed as previously described (Corrêa et al., 2007). Briefly, plasma samples (1.0 ml) were extracted using the acetone/petroleum ether method, lyophilized and stored at −70 °C until analysis. Standard reagents and incubation protocols were used

54

T.F. Martins et al. / Journal of Neuroimmunology 238 (2011) 52–57

for the peptide assays. AVP measurements were undertaken with a commercial antiserum (Peninsula Laboratories) at a final dilution of 1:20,000. The antiserum is specific and shows essentially no crossreactivity with other known peptides. The buffer used composed of Na2HPO4 (0.062 M) and Na2EDTA (0.013 M) supplemented with 0.5% BSA, pH 7.5. For peptide labeling, 125I was purchased from a commercial supplier (Amersham). A non-equilibrium assay was used with an incubation volume of 500 μl and an incubation time of 4 days at 4 °C. Bound hormone was separated from unbound by a secondary antibody produced in the laboratory of José AntunesRodrigues and Lucila L.K. Elias where the RIA was performed. The minimum detection limit was 0.9 pg/ml and the coefficients of interand intra-assay variations were 11% and 7%, respectively. 2.7. Data analysis Survival rate was expressed as percentage, and a Log-rank (Mantel– Cox) test was used to determine differences in survival curves. The other data are reported as mean ± S.E.M. Statistical analyses were performed by two-way analysis of variance (ANOVA) and a post hoc Tukey test. Values of P b 0.05 were considered statistically significant. RIA data were analyzed by logit transformation of the raw data.

Fig. 2. Effect of intraperitoneal administration of MK-886 on neutrophil migration into the peritoneal cavity (left panel) 4 h after CLP. The results are expressed as means ± SEM from 4 rats in each group.

Plasma protein levels of the CLP group were decreased at 4 and 24 h after surgery. Concerning serum sodium and plasma osmolality we observed minor alterations during sepsis. Pretreatment with MK-886 did not substantially affect the changes observed in these parameters.

3. Results 3.4. Effects of MK-886 on serum nitrate 3.1. Effects of MK-886 on survival rate The survival rate in the animals submitted to CLP was 45% in the first day and 20% in the second and third day after surgery. Overall, the injection of MK-886 did not significantly alter survival (Fig. 1). In the sham groups (sham + DMSO 5% or sham + MK-886), all rats survived for 3 days after surgery (data not shown). 3.2. Effects of MK-886 on neutrophil migration Concerning neutrophil migration into the peritoneal cavity we did not detect any alteration in this parameter at the 4 h time point, independent of MK-886 pretreatment or not. The neutrophil measurement is indicative that the degree of sepsis was similar in the MK-886 treated and untreated CLP rats (Fig. 2).

We observed an increase on serum nitrate at 4 and 24 h after CLP. Pretreatment with MK-886 seemed to abolish the CLP-induced NO increase seen at 4 h in a dose-dependent manner, but this was not validated through statistical analysis (Fig. 3). 3.5. Effects of MK-886 in the plasma AVP levels Sepsis induced by CLP, induced a significant increase in plasma AVP levels at 4 h after surgery. At 24 h the concentrations had dropped to basal. Pretreatment with MK-886 in the doses of 1.0 and 2.0 mg/kg attenuated the release of the hormone. The dose of 4.0 mg/ kg of MK-886 completely abolished this hormonal response in the early phase of sepsis (Fig. 4). 4. Discussion

3.3. Effects of MK-886 on hematocrit, serum sodium and nitrate, plasma protein and osmolality In the CLP group we observed at 4 h an increase in hematocrit followed by a return to basal levels at 24 h after surgery (Table 1).

Fig. 1. Effect of intraperitoneal administration of MK-886 on survival rate. Number of animals in parentheses.

The CLP experimental model was chosen because it best reproduces the complex immunopathology of sepsis and closely resembles the clinical situation of peritonitis due to mixed intestinal microbiota (Echtenacher et al., 2001; Freise et al., 2001; Rios-Santos et al., 2003; Nemzek et al., 2004). All our CLP animals developed the clinical signs of sepsis, including lethargy, piloerection and diarrhea, whereas sham animals remained active in their cages. The survival rate was lower in the CLP animals compared with sham rats, and no survival advantage was afforded by the LTs synthesis inhibitor. In our experiments, MK-886 also did not modify neutrophil migration into the peritoneal cavity in rats submitted to CLP. These results are in agreement with experiments done in lethalCLP (puncturing 14 times with a 21-gage needle) (Rios-Santos et al., 2003) and studies that also did not reveal any alteration in survival rate in LPS-induced sepsis (Azab and Kaplanski, 2004). In the present study, we set the focus on the role of LTs in the modulation of AVP release during experimental sepsis. Additionally we asked whether this modulation could be through NO production because the LTs seem to affect the production of this mediator (Wilson et al., 2001; Knigge et al., 2003; Won et al., 2005). During the initial phase of sepsis, CLP produced a significant increase in plasma AVP levels at 4 h, which returned to basal ones at 24 h, as shown in previous studies (Pancoto et al., 2008; Athayde et al., 2009). Intraperitoneal injection of MK-886, a LTs synthesis inhibitor,

T.F. Martins et al. / Journal of Neuroimmunology 238 (2011) 52–57

55

Table 1 Effect of intraperitoneal administration of MK-886 on hematocrit, plasma protein, serum sodium and osmolality. DMSO 5% Sham Hematocrit 0 4 24

(%) 43.9 ± 1.0 45.7 ± 1.8 43.4 ± 1.4

Protein (g/dl) 0 11.4 ± 0.4 4 8.4 ± 0.9a 24 10.7 ± 0.5

MK-886 (1 mg/kg) CLP

Sham

MK-886 (2 mg/kg) CLP

Sham

MK-886 (4 mg/kg) CLP

Sham

CLP

46.5 ± 0.8 56.3 ± 1.3a 46.7 ± 1.0

44.3 ± 0.5 45.5 ± 0.8 43.6 ± 1.8

44.2 ± 1.3 52.4 ± 2.3a 47.6 ± 1.1

44.8 ± 0.8 47.4 ± 1.4 44.4 ± 0.5

44.1 ± 0.8 53.5 ± 0.9a 46.4 ± 1.5

44.7 ± 1.0 47.1 ± 1.2 44.4 ± 0.8

44.7 ± 1.0 54.1 ± 1.7a 46.7 ± 2.0

11.4 ± 0.3 8.4 ± 0.4b 8.1 ± 0.5b,c

10.1 ± 0.6 10.0 ± 0.5 10.5 ± 0.4

9.7 ± 0.3 9.4 ± 0.7 8.2 ± 0.8c

11.2 ± 0.1 9.4 ± 0.5 9.9 ± 0.2

10.9 ± 0.3 7.6 ± 0.6b,c 7.4 ± 0.0b,c

11.4 ± 0.2 8.8 ± 0.5a 11.4 ± 0.4

10.7 ± 0.4 8.4 ± 0.4 7.9 ± 0.5b,c

Sodium (meq/kg) 0 136.0 ± 1.8 4 140.8 ± 1.1 24 136.6 ± 1.4

140.4 ± 1.4 141.0 ± 1.8 135.6 ± 1.3a

137.8 ± 1.9 140.2 ± 2.2 139.5 ± 0.5

142.3 ± 1.1 138.8 ± 1.5 135.3 ± 1.7b

140.6 ± 0.8 139.0 ± 1.5 141.8 ± 1.2

142.8 ± 1.2 135.8 ± 1.3b 139.0 ± 1.8

141.6 ± 1.0 140.8 ± 1.1 139.6 ± 1.4

141.4 ± 1.2 136.8 ± 1.8 138.2 ± 1.9

Osmolality 0 4 24

284.0 ± 2.5 274.6 ± 2.2b 266.6 ± 5.2b

279.5 ± 3.0 278.8 ± 4.4 265.2 ± 3.9a

277.0 ± 3.0 277.5 ± 2.4 258.8 ± 3.4a

291.4 ± 3.3 276.2 ± 2.1b 274.0 ± 2.1b

285.7 ± 4.5 271.0 ± 3.2b 265.0 ± 3.4b

288.2 ± 2.2 274.0 ± 2.4b 277.9 ± 1.4b

288.7 ± 3.0 271.5 ± 1.3b 263.7 ± 3.1b

(mOsm/kg) 281.7 ± 2.8 270.1 ± 2.7b 274.3 ± 3.5b

Measurements are expressed as mean ± SEM for each group comprising 5–10 animals. CLP, cecal ligation and puncture. a P b 0.05 compared to 0 and 4 h within the group. b P b 0.05 compared to 0 h within the group. c P b 0.05 compared to the sham group.

attenuated or prevented this increase in a dose-dependent manner. We hypothesize that its effect is independent of NO production, because though the i.p. injection of MK-886 seems to prevent the increase in nitrate production that usually occurs at 4 h after CLP, overall there was no significant difference between the groups pretreated or not with the drug. Increase in serum sodium levels and osmolality are strong stimuli for AVP release. We herein observed a significant reduction in sodium and osmolality at 4 and 24 h after CLP surgery. In previous studies we observed a progressive increase in water intake at 4 h after CLP but no alteration in diuresis (Corrêa et al., 2007; Oliveira-Pelegrin et al., 2009), indicating a dilution of the body fluid and consequent reduction in serum sodium and osmolality. However, none of these parameters were altered by pretreatment with MK-886. Hypovolemia that usually occurs during sepsis may also contribute to the observed AVP release (Parrillo, 1993). Accordingly we could see an increase in hematocrit at 4 h of sepsis coinciding with the increase in plasma AVP levels and in some time points with a drop in plasma protein. However, the pretreatment with MK-886 did not substan-

Fig. 3. Effect of intraperitoneal administration of MK-886 on serum nitrate. The results are expressed as means ± SEM. §P b 0.05 compared to 0 h within the group. ⁎P b 0.001 compared to 0 and 4 h within the group. n = 6–10 each group.

tially change plasma protein and hematocrit levels. Hypovolemia can be due to the liberation of inflammatory mediators that can cause damage to endothelial cell, with consequent capillary leakage and major loss of fluid and protein into the interstitial space (Parrillo, 1993; Andrew et al., 2000; Fishel et al., 2003; Benjamim et al., 2005). In a previous study we already observed a decrease in plasma protein concentration after CLP surgery that was attenuated following pretreatment with aminoguanidine, an iNOS inhibitor (Corrêa et al., 2007). Several explanations are possible for the effect of MK-886 on the liberation of AVP during the initial phase of the sepsis. An attenuation of the increase in the plasma concentration of this hormone was also observed when we systemically inhibited NO production by administration of aminoguanidine (Corrêa et al., 2007). We concluded that the decrease in blood pressure due to the liberation of NO and other mediators can be at least partially responsible for the AVP release observed in the early phase of sepsis. Similarly, the fact that i.c.v injection of MK-886 attenuated and even abolished the increase in the

Fig. 4. Effect of intraperitoneal administration of MK-886 on plasma vasopressin levels. The results are expressed as means ± SEM; §P b 0.05 compared to 4 h within the group; ⁎P b 0.05 compared to CLP + DMSO group; #P b 0.05 compared to CLP + 1 mg/kg group; † P b 0.05 compared to CLP + 2 mg/kg group. n = 6–12 each group.

56

T.F. Martins et al. / Journal of Neuroimmunology 238 (2011) 52–57

AVP plasma levels could be explained as resulting from an observed increase or normalization of the blood pressure (Benjamim et al., 2005; Athayde et al., 2009). Reestablishment of blood pressure was also seen in mice pretreated with MK-571 a cys-LTs receptor antagonist before being submitted to CLP (Benjamim et al., 2005). It is possible that the i.p. injection of MK-886 acted centrally because LTC4 synthase, but not LTA4 hydrolase, is selectively localized in vasopressinergic neurons of the hypothalamus (Shimada et al., 2005). Moreover together with its vehicle it can cross the blood–brain barrier (Iwen and Miller, 1986), and thus be directly responsible for the diminished AVP release. Previous studies showed that i.p injection of MK-886 attenuated the increase in LPS-induced production of hypothalamic cys-LTs (Paul et al., 1999; Azab and Kaplanski, 2004), suggesting that the drug can reach the central nervous system. Furthermore, we could previously show that the i.c.v injection of MK886 blocked the increase in hypothalamic LTC4 synthase observed 4 h after CLP. This effect happened at the same time as the attenuated increase in plasma AVP levels. These results, therefore, indicate a role of central LTs in the liberation of AVP. The present study now showed that the MK-886 administered intraperitoneally is also able to decrease the vasopressin release in a dose–response manner during sepsis, implicating a role for leukotrienes. Additionally this effect seems to be independent of NO production.

Acknowledgments The authors thank Nadir M. Fernandes, Marina Holanda, Milene M. Lopes, Sônia A. Zanon and Maria Valci Silva for the excellent technical assistance and Adriana Moreno for the help with the figures' artwork. We also thank Drs. José Antunes-Rodrigues and Lucila L. K. Elias from the Departmento de Fisiologia, Faculdade de Medicina de Ribeirão Preto, USP, for providing the infrastructure for the RIA analyses. Merck Frost Canada Inc kindly provided the MK-886. Financial support from FAPESP and CAPES are gratefully acknowledged.

References Andrew, P., Deng, Y., Kaufman, S., 2000. Fluid extravasation from spleen reduces blood volume in endotoxemia. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278, R60–R65. Athayde, L.A., Oliveira-Pelegrin, G.R., Nomizo, A., Faccioli, L.H., Rocha, M.J., 2009. Blocking central leukotrienes synthesis affects vasopressin release during sepsis. Neuroscience 160, 829–836. Azab, A.N., Kaplanski, J., 2004. Involvement of eicosanoids in the hypothermic response to lipopolysaccharide during endotoxemia in rats. Prostaglandins Leukot. Essent. Fatty Acids 70, 67–75. Baenkler, M., Leykauf, M., John, S., 2006. Functional analysis of eicosanoids from white blood cells in sepsis and SIRS. J. Physiol. Pharmacol. 57 (Suppl. 12), 25–33. Benjamim, C.F., Canetti, C., Cunha, F.Q., Kunkel, S.L., Peters-Golden, M., 2005. Opposing and hierarchical roles of leukotrienes in local innate immune versus vascular responses in a model of sepsis. J. Immunol. 174, 1616–1620. Bisgaard, H., Loland, L., Oj, J.A., 1999. NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast. Am. J. Respir. Crit. Care Med. 160, 1227–1231. Bratton, D.L., Lanz, M.J., Miyazawa, N., White, C.W., Silkoff, P.E., 1999. Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: a preliminary study. Pediatr. Pulmonol. 28, 402–407. Carnio, E.C., Moreto, V., Giusti-Paiva, A., Antunes-Rodrigues, J., 2006. Neuro-immuneendocrine mechanisms during septic shock: role for nitric oxide in vasopressin and oxytocin release. Endocr. Metab. Immune Disord. Drug Targets 6, 137–142. Clark, J.D., Schievella, A.R., Nalefski, E.A., Lin, L.L., 1995. Cytosolic phospholipase A2. J. Lipid Mediat. Cell Signal. 12, 83–117. Corrêa, P.B., Pancoto, J.A., de Oliveira-Pelegrin, G.R., Carnio, E.C., Rocha, M.J., 2007. Participation of iNOS-derived NO in hypothalamic activation and vasopressin release during polymicrobial sepsis. J. Neuroimmunol. 183, 17–25. Cunningham, F.M., Shipley, M.E., Smith, M.J., 1980. Aggregation of rat polymorphonuclear leucocytes in vitro. J. Pharm. Pharmacol. 32, 377–380. Dahlen, S.E., Hedqvist, P., Hammarstrom, S., Samuelsson, B., 1980. Leukotrienes are potent constrictors of human bronchi. Nature 288, 484–486. Di Gennaro, A., Carnini, C., Buccellati, C., Ballerio, R., Zarini, S., Fumagalli, F., Viappiani, S., Librizzi, L., Hernandez, A., Murphy, R.C., Constantin, G., De Curtis, M., Folco, G., Sala, A., 2004. Cysteinyl-leukotrienes receptor activation in brain inflammatory reactions and cerebral edema formation: a role for transcellular biosynthesis of cysteinylleukotrienes. FASEB J. 18, 842–844.

Echtenacher, B., Freudenberg, M.A., Jack, R.S., Mannel, D.N., 2001. Differences in innate defense mechanisms in endotoxemia and polymicrobial septic peritonitis. Infect. Immun. 69, 7271–7276. Fishel, R.S., Are, C., Barbul, A., 2003. Vessel injury and capillary leak. Crit. Care Med. 31, S502–S511. Flamand, N., Mancuso, P., Serezani, C.H., Brock, T.G., 2007. Leukotrienes: mediators that have been typecast as villains. Cell Mol. Life Sci. 64, 2657–2670. Ford-Hutchinson, A.W., Bray, M.A., Doig, M.V., Shipley, M.E., Smith, M.J., 1980. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286, 264–265. Freise, H., Bruckner, U.B., Spiegel, H.U., 2001. Animal models of sepsis. J. Invest. Surg. 14, 195–212. Friedman, B.S., Bel, E.H., Buntinx, A., Tanaka, W., Han, Y.H., Shingo, S., Spector, R., Sterk, P., 1993. Oral leukotriene inhibitor (MK-886) blocks allergen-induced airway responses. Am. Rev. Respir. Dis. 147, 839–844. Gimbrone Jr., M.A., Brock, A.F., Schafer, A.I., 1984. Leukotriene B4 stimulates polymorphonuclear leukocyte adhesion to cultured vascular endothelial cells. J. Clin. Invest. 74, 1552–1555. Goulet, J.L., Byrum, R.S., Key, M.L., Nguyen, M., Wagoner, V.A., Koller, B.H., 2000. Genetic factors determine the contribution of leukotrienes to acute inflammatory responses. J. Immunol. 164, 4899–4907. Heise, C.E., O'Dowd, B.F., Figueroa, D.J., Sawyer, N., Nguyen, T., Im, D.S., Stocco, R., Bellefeuille, J.N., Abramovitz, M., Cheng, R., Williams Jr., D.L., Zeng, Z., Liu, Q., Ma, L., Clements, M.K., Coulombe, N., Liu, Y., Austin, C.P., George, S.R., O'Neill, G.P., Metters, K.M., Lynch, K.R., Evans, J.F., 2000. Characterization of the human cysteinyl leukotriene 2 receptor. J. Biol. Chem. 275, 30531–30536. Henderson, W.R., Klebanoff, S.J., 1983. Leukotriene production and inactivation by normal, chronic granulomatous disease and myeloperoxidase-deficient neutrophils. J. Biol. Chem. 258, 13522–13527. Hirabayashi, T., Shimizu, T., 2000. Localization and regulation of cytosolic phospholipase A(2). Biochim. Biophys. Acta 1488, 124–138. Holmes, C.L., Patel, B.M., Russell, J.A., Walley, K.R., 2001. Physiology of vasopressin relevant to management of septic shock. Chest 120, 989–1002. Hulting, A.L., Lindgren, J.A., Hokfelt, T., Eneroth, P., Werner, S., Patrono, C., Samuelsson, B., 1985. Leukotriene C4 as a mediator of luteinizing hormone release from rat anterior pituitary cells. Proc. Natl. Acad. Sci. U. S. A. 82, 3834–3838. Iwen, P.C., Miller, N.G., 1986. Enhancement of ketoconazole penetration across the blood–brain barrier of mice by dimethyl sulfoxide. Antimicrob. Agents Chemother. 30, 617–618. Knigge, U., Kjaer, A., Kristoffersen, U., Madsen, K., Toftegaard, C., Jorgensen, H., Warberg, J., 2003. Histamine and prostaglandin interaction in regulation of oxytocin and vasopressin secretion. J. Neuroendocrinol. 15, 940–945. Lammers, C.H., Schweitzer, P., Facchinetti, P., Arrang, J.M., Madamba, S.G., Siggins, G.R., Piomelli, D., 1996. Arachidonate 5-lipoxygenase and its activating protein: prominent hippocampal expression and role in somatostatin signaling. J. Neurochem. 66, 147–152. Landry, D.W., Oliver, J.A., 2001. The pathogenesis of vasodilatory shock. N. Engl. J. Med. 345, 588–595. Landry, D.W., Levin, H.R., Gallant, E.M., Ashton Jr., R.C., Seo, S., D'Alessandro, D., Oz, M.C., Oliver, J.A., 1997. Vasopressin deficiency contributes to the vasodilation of septic shock. Circulation 95, 1122–1125. Lindgren, J.A., Hulting, A.L., Hokfelt, T., Dahlen, S.E., Eneroth, P., Werner, S., Patrono, C., Samuelsson, B., 1985. Evidence for leukotriene formation and a neuroendocrine role for leukotriene C4 in rat brain. Adv. Prostaglandin Thromboxane Leukot. Res. 15, 561–564. Maxime, V., Siami, S., Annane, D., 2007. Metabolism modulators in sepsis: the abnormal pituitary response. Crit. Care Med. 35, S596–S601. Morlion, B.J., Torwesten, E., Kuhn, K.S., Puchstein, C., Furst, P., 2000. Cysteinylleukotriene generation as a biomarker for survival in the critically ill. Crit. Care Med. 28, 3655–3658. Murphy, R.C., Gijon, M.A., 2007. Biosynthesis and metabolism of leukotrienes. Biochem. J. 405, 379–395. Nemzek, J.A., Xiao, H.Y., Minard, A.E., Bolgos, G.L., Remick, D.G., 2004. Humane endpoints in shock research. Shock 21, 17–25. Oliveira-Pelegrin, G.R., Ravanelli, M.I., Branco, L.G., Rocha, M.J., 2009. Thermoregulation and vasopressin secretion during polymicrobial sepsis. Neuroimmunomodulation 16, 45–53. Pancoto, J.A., Correa, P.B., Oliveira-Pelegrin, G.R., Rocha, M.J., 2008. Autonomic dysfunction in experimental sepsis induced by cecal ligation and puncture. Auton. Neurosci. 138, 57–63. Parrillo, J.E., 1993. Pathogenetic mechanisms of septic shock. N. Engl. J. Med. 328, 1471–1477. Paul, L., Fraifeld, V., Kaplanski, J., 1999. Evidence supporting involvement of leukotrienes in LPS-induced hypothermia in mice. Am. J. Physiol. 276, R52–R58. Peters-Golden, M., Brock, T.G., 2003. 5-lipoxygenase and FLAP. Prostaglandins Leukot. Essent. Fatty Acids 69, 99–109. Peters-Golden, M., Henderson Jr., W.R., 2007. Leukotrienes. N. Engl. J. Med. 357, 1841–1854. Rios-Santos, F., Benjamim, C.F., Zavery, D., Ferreira, S.H., Cunha Fde, Q., 2003. A critical role of leukotriene B4 in neutrophil migration to infectious focus in cecal ligation and puncture sepsis. Shock 19, 61–65. Sandrini, A., Ferreira, I.M., Gutierrez, C., Jardim, J.R., Zamel, N., Chapman, K.R., 2003. Effect of montelukast on exhaled nitric oxide and nonvolatile markers of inflammation in mild asthma. Chest 124, 1334–1340. Sharshar, T., Blanchard, A., Paillard, M., Raphael, J.C., Gajdos, P., Annane, D., 2003. Circulating vasopressin levels in septic shock. Crit. Care Med. 31, 1752–1758.

T.F. Martins et al. / Journal of Neuroimmunology 238 (2011) 52–57 Shimada, A., Satoh, M., Chiba, Y., Saitoh, Y., Kawamura, N., Keino, H., Hosokawa, M., Shimizu, T., 2005. Highly selective localization of leukotriene C4 synthase in hypothalamic and extrahypothalamic vasopressin systems of mouse brain. Neuroscience 131, 683–689. Smedegard, G., Hedqvist, P., Dahlen, S.E., Revenas, B., Hammarstrom, S., Samuelsson, B., 1982. Leukotriene C4 affects pulmonary and cardiovascular dynamics in monkey. Nature 295, 327–329. Sprague, R.S., Stephenson, A.H., Dahms, T.E., Lonigro, A.J., 1989. Proposed role for leukotrienes in the pathophysiology of multiple systems organ failure. Crit. Care Clin. 5, 315–329.

57

Sriskandan, S., Altmann, D.M., 2008. The immunology of sepsis. J. Pathol. 214, 211–223. Wilson, A.M., Dempsey, O.J., Sims, E.J., Lipworth, B.J., 2001. A comparison of topical budesonide and oral montelukast in seasonal allergic rhinitis and asthma. Clin. Exp. Allergy 31, 616–624. Won, J.S., Im, Y.B., Khan, M., Singh, A.K., Singh, I., 2005. Involvement of phospholipase A2 and lipoxygenase in lipopolysaccharide-induced inducible nitric oxide synthase expression in glial cells. Glia 51, 13–21.