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Originally published online as doi:10.1189/jlb.0407217 on June 12, 2007

Published online before print June 12, 2007
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(Journal of Leukocyte Biology. 2007;82:678-685.)
© 2007 by Society for Leukocyte Biology

Neurokinin-1 receptor antagonist treatment protects mice against lung injury in polymicrobial sepsis

Akhil Hegde*, Huili Zhang*, Shabbir M. Moochhala*,{dagger} and Madhav Bhatia*,1

* Cardiovascular Biology Program, Department of Pharmacology, National University of Singapore, Singapore; and
{dagger} Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore

1 Correspondence: A/P Madhav Bhatia, Centre for Life Sciences, #03-06, 28 Medical Drive, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117456. E-mail: mbhatia{at}nus.edu.sg


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Earlier work from our laboratory has suggested a role for the neuropeptide substance P (SP) in inducing lung injury in sepsis. In that study, mice lacking the preprotachykinin-A gene, which encodes for SP, were protected against lung injury in sepsis. To further substantiate the role of SP in sepsis and to study its mechanism, we have evaluated the effect of SR140333, a SP receptor antagonist, on lung injury in sepsis, which was induced in male Swiss mice by cecal ligation and puncture (CLP). Sham-operated animals received the same surgical procedure, except CLP. Vehicle or SR140333 (1 mg/kg, s.c.) was administered to CLP mice 30 min before or 1 h after the CLP. Eight hours after surgery, lung tissue was collected and analyzed for myeloperoxidase (MPO) activity, chemokines, cytokines, and adhesion molecules. The CLP procedure alone caused a significant increase in the lung levels of MIP-2, MCP-1, IL-1ß, IL-6, ICAM-1, E- and P-selectin, and MPO activity when compared with sham-operated mice. SR140333 injected 30 min before or 1 h after CLP significantly attenuated the increased lung MPO activity and levels of MIP-2, MCP-1, IL-1ß, IL-6, ICAM-1, and E- and P-selectin compared with CLP-operated mice injected with the vehicle. Histological evaluation of the lung sections further supported the beneficial effect of SR140333 on lung inflammation. Therefore, SP receptor antagonism can be a potential therapeutic target in polymicrobial sepsis, and this effect is brought about via reduction in leukocyte recruitment.

Key Words: SR140333 • cecal ligation and puncture • substance P • NK-1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients with burn, hemorrhage, traumatic injury, or abdominal surgery are highly vulnerable to pathogens and opportunistic infections. In addition, critically ill, old, pediatric, and postoperational patients are susceptible to these infections. A minor wound infection in these patients can easily end up in polymicrobial sepsis [1 ]. In the United States, for instance, ~750,000 people develop sepsis annually and one-third of them die of the resulting multiple organ failure [2 ]. Sepsis has a high mortality rate, especially in immunocompromised conditions [1 ]. Predominantly supportive treatment, with no effective therapies so far, does not help in reducing the death rate of sepsis [3 ].

Sepsis is the intense systemic inflammatory response syndrome, caused usually by bacterial infection [4 ]. When the immune defenses of the body fail to eliminate pathogens, infection spreads through blood circulation. The resultant production of proinflammatory cytokines and chemokines leads to recruitment of neutrophils, tissue damage, and multiple organ failure [3 ]. However, it is the subsequent excessive production of anti-inflammatory mediators that induces immunosuppression and fatalities in sepsis [4 , 5 ]. The balance between proinflammatory and anti-inflammatory mediators plays an important role in the pathophysiology of sepsis.

Substance P (SP), a preprotachykinin-A (PPT-A) gene product, is an immunoregulatory neuropeptide produced at various inflammation sites. It is found in resident macrophages and circulating leukocytes [6 , 7 ] and is known to have a role in neurogenic inflammation [8 ]. The tachykinin SP is reported to increase postcapillary venule permeability, immune cell influx, and glandular secretion in mammalian airways [9 ]. SP binds to neurokinin-1 (NK-1) G protein-coupled receptors on the surface of effector cells and acts as a proinflammatory mediator in many inflammatory states [7 , 10 ]. NK-1 receptor activation has been shown to enhance inflammation by decreasing the vascular tone and increasing the endothelial microvascular permeability and transport of inflammatory cells [8 ]. Increased SP immunoreactivity has been found in bronchoalveolar lavage samples from patients suffering from lung diseases [11 ]. SP and NK-1 receptor have been implicated in the up-regulation of ICAM-1 on vascular endothelial cells and neutrophil infiltration [12 ] and leukocyte adhesion to the endothelial or epithelial cells in the airways [13 , 14 ] in inflammation. It is interesting that PPT-A knockout mice are reported to be protected against acute pancreatitis and associated lung injury [10 ]. Recently, our group has shown that PPT-A knockout mice are protected against polymicrobial sepsis [15 ].

In addition to the use of gene knockout animal models, it was imperative to block the receptors pharmacologically to understand the mechanism of action of SP in sepsis. SR140333 (nolpitantium) is a highly potent and selective antagonist of the tachykinin NK-1 receptors in humans and other animals [16 ]. It has been shown to reduce the severity of inflammation in trinitrobenzene sulfonic acid-induced colitis in the rat colon [17 ]. SR140333 inhibited mustard oil-induced plasma protein extravasations in the dorsal skin of the rat hind paw [18 ]. It is also reported to reduce arachidonate release from alveolar macrophages in guinea pigs exposed to SP [19 ]. Recently, SR140333 was found to be effective in the modulation of the inflammatory response and airway remodeling in mice [20 ]. Further, SR140333 is reported to cause antagonism of the SP-induced relaxations of human isolated intralobar pulmonary arterial rings [21 ].

Therefore, the present study was aimed at evaluating the role of SP in polymicrobial sepsis in mice. In bowel perforation-induced peritonitis patients, infection results from a mixed intestinal flora [22 ]. Cecal ligation and puncture (CLP) is a similar polymicrobial sepsis model, which is reliable and more clinically relevant. Thus, we used CLP to cause polymicrobial sepsis in mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal model of polymicrobial sepsis
All animal experiments performed were in accordance with the guidelines of the Defence Science Organisation Animal Care and Use Committee, Defence Medical and Environmental Research Institute (Singapore), which follows the established International Guiding Principles for Animal Research. Swiss male mice (25–30 g) used for the study were assigned randomly to sham or CLP experimental groups (n>6 in each group). Polymicrobial sepsis was induced in mice by CLP [23 , 24 ]. Following strict aseptic conditions, the anterior abdomen was shaved, and a midline incision was made in the lower part of the abdomen. The peritoneum was opened, and the cecum was ligated below the ileocecal valve with 4/0 silk suture without obstructing the bowel. The cecum was punctured twice with a 22-gauge needle and squeezed gently to extrude the cecal contents. The cecum was placed back in the abdomen, and the muscle and skin incision was sutured separately. All the mice were given saline (1 ml, s.c) after the surgery and kept on heat pads for recovery. The same surgical procedure except the CLP was performed on sham-operated animals. Vehicle (DMSO diluted in PBS, 0.25% v/v) or SR140333 (1 mg/kg; 0.25 mg/ml, s.c.) was administered to CLP-operated mice 30 min before (pretreatment) or 1 h after (post-treatment) the CLP (SR140333 was provided by Sanofi Synthelabo, France).

The animals were killed 8 h after surgery by an i.p. injection of a lethal dose of pentobarbitone. Blood was collected by cardiac puncture, heparinized, and centrifuged and plasma removed and stored at –80°C. Samples of lung were snap-frozen in liquid nitrogen and stored at –80°C for subsequent measurement of tissue myeloperoxidase (MPO) activity and chemokine, cytokine, and adhesion molecule levels. Random cross-sections of lung were fixed in 4% neutral phosphate-buffered formalin and embedded in paraffin wax.

Another set of animals was divided randomly into sham or CLP experimental groups (n=6 in each group). Polymicrobial sepsis was induced in CLP experimental mice by CLP [23 , 24 ]. The same surgical procedure, except the CLP was performed on sham-operated animals. Vehicle (saline) or L703606 (4 mg/kg; 1 mg/ml, i.p.) was administered to CLP-operated mice 30 min before (pretreatment) the CLP (L703606, a potent and selective, nonpeptide NK-1 receptor antagonist, was purchased from Sigma-Aldrich, Singapore). The animals were killed 8 h after surgery, and lung samples were snap-frozen in liquid nitrogen and stored at –80°C for subsequent measurement of MPO activity.

MPO estimation
MPO activity as a measure of neutrophil sequestration in lung was quantified as described previously [10 , 25 , 26 ]. Tissue samples were thawed, homogenized in 20 mM phosphate buffer (pH 7.4), and centrifuged (10,000 g, 10 min, 4°C), and the resulting pellet was resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma Chemical Co., St. Louis, MO, USA). The suspension was subject to four cycles of freezing and thawing and further disrupted by sonication (40 s). The sample was then centrifuged (10,000 g, 5 min, 4°C), and the supernatant was used for the MPO assay. The reaction mixture consisted of the supernatant, 1.6 mM tetramethylbenzidine (Sigma Chemical Co.), 80 mM sodium phosphate buffer (pH 5.4), and 0.3 mM hydrogen peroxide. This mixture was incubated at 37°C for 110 s, the reaction terminated with 2 M H2SO4, and the absorbance measured at 450 nm. The absorbance was then corrected for the DNA content of the tissue sample [27 ]. Results were expressed as fold increase over control.

Histopathological examination
Paraffin-embedded lung samples were sectioned at 5 µm thickness, stained with H&E, and evaluated by light microscopy and documented by photographs. Eight randomly chosen microscopic fields (x125) were examined for each tissue sample, and the extent of cell injury/necrosis, represented by the destruction of histoarchitecture of the cells, vacuolization, and swelling of cells, all of which have been associated with an inflammatory reaction, was evaluated.

ELISA analysis of chemokines, cytokines, and adhesion molecules
Plasma and tissue homogenates were assayed to evaluate the level of chemokines (MCP-1, MIP-2, and RANTES), cytokines (IL-6, IL-1ß, and TNF-{alpha}), and adhesion molecules (E- and P-selectins, ICAM-1, and VCAM-1) by a sandwich ELISA, according to the manufacturer’s instructions. DuoSet ELISA kits with matched antibody pairs against mouse chemokine/cytokine/adhesion molecule were obtained from R&D Systems (Minneapolis, MN, USA). Briefly, an antichemokine/cytokine/adhesion molecule primary antibody was coated onto 96-well ELISA plates and incubated overnight at room temperature. Samples and standards were added to the wells and incubated for 2 h, the wells were washed, and a biotinylated goat anti-mouse chemokine/cytokine/adhesion molecule antibody was added for 2 h. Plates were washed again, and streptavidin conjugated to HRP was added for 20 min. After a further wash, tetramethylbenzidine was added for color development, and the reaction was terminated with 2 N H2SO4. Absorbance was measured at 450 nm. Sample concentration was estimated from the standard curve. DNA assay was performed fluorometrically by using Hoechst dye 33256 by the method of Labarca and Paigen [27 ]. The sample concentration was then corrected for the DNA content of the tissue [27 ].

Statistical analysis
All values were expressed as mean ± SEM. The significance of changes was evaluated by using ANOVA when comparing three or more groups and Tukey’s method as a post hoc test for comparison among different groups. A P value of <0.05 was considered to indicate a significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of SR140333 treatment on neutrophil sequestration in lung in CLP mice
Neutrophil infiltration was quantified by measuring tissue MPO activity. Increased MPO activities represent recruitment of neutrophils and a state of inflammation. Following 8 h after CLP, MPO activity in the lung was increased significantly in vehicle treated in pre- and post-CLP animals when compared with the sham mice (Fig. 1A ). Treatment with SR140333, 30 min before or 1 h after CLP, reduced the MPO activity significantly in the lung (Fig. 1A) . Similarly, administration of another NK-1 receptor antagonist, L703606, 30 min before CLP reduced the lung MPO activity significantly (Fig. 1B) .


Figure 1
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  		Figure 1. (A) Effect of SR140333 administration 30 min before or 1 h after CLP on lung neutrophil infiltration. Mice (n=6–9 in each group) were divided into CLP-operated and sham-operated groups. CLP-operated mice received vehicle (DMSO in PBS, 0.25% v/v) or SR140333 (1 mg/kg; 0.25 mg/ml) s.c. 30 min before (pretreatment) or 1 h after (post-treatment) the CLP. (B) Effect of L703606 administration 30 min before CLP on lung neutrophil infiltration. Mice (n=6 in each group) were divided into CLP-operated and sham-operated groups. CLP-operated mice received vehicle (saline) or L703606 (4 mg/kg; 1 mg/ml) i.p. 30 min before (pretreatment) the CLP. The same surgical procedure as the CLP-operated animals, except CLP was performed on sham-operated animals. Eight hours after the CLP procedure, mice were killed, and lung MPO activity was determined as described in Materials and Methods. Results shown are the mean ± SEM. *, P < 0.001, when vehicle-treated, CLP animals were compared with sham group animals; **, P < 0.05, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals; ***, P < 0.001, when L703606-treated, CLP animals were compared with vehicle-treated, CLP animals.

 
Figure 2A 2B 2C 2D 2E , shows representative, H&E-stained lung sections from sham and CLP-operated mice. Histological evaluation of lung sections showed a significant increase in alveolar thickening, an indicator of edema, as well as inflammatory infiltration in CLP animals treated only with vehicle (Fig. 2B and 2D) . Lung section from the sham group showed little or no edema and inflammatory infiltration (Fig. 2A) . Prophylactic and therapeutic treatment with SR140333 reduced the lung injury significantly, represented by reduced lung edema and neutrophil infiltration (Fig. 2C and 2E) .


Figure 2
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  		Figure 2. Morphological changes in H&E-stained mouse lung on induction of sepsis. (A) Sham: no CLP; (B) vehicle (DMSO in PBS, 0.25% v/v) administered 30 min before CLP-pretreatment control; (C) SR140333 (1 mg/kg) administered 30 min before CLP-SR140333 pretreatment; (D) vehicle (DMSO in PBS, 0.25% v/v) administered 1 h after CLP-post-treatment control; (E) SR140333 (1 mg/kg) administered 1 h after CLP-SR140333 post-treatment.

 
Effect of SR140333 treatment on chemokine levels in lung
Chemokines are produced in response to infection. They act as chemoattractants to various inflammatory cells in sepsis. We determined the levels of CXC chemokine MIP-2 and CC chemokines MCP-1 and RANTES in lung tissue. As expected, lung MIP-2 levels in CLP mice without SR140333 treatment were significantly higher compared with the sham levels (Fig. 3A ). Administration of SR140333, pre- and post-CLP, resulted in a significant decrease in lung MIP-2 levels compared with the corresponding levels in vehicle-treated, CLP mice (Fig. 3A) . The CLP procedure increased the production of lung RANTES slightly compared with that in sham animals, and the increase was significant in animals treated with vehicle 1 h after CLP (Fig. 3B) . However, SR140333, when injected 30 min or 1 h after CLP, resulted in a significant decrease in lung RANTES levels compared with the corresponding levels in the absence of SR140333 (Fig. 3B) .


Figure 3
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  		Figure 3. Effect of SR140333 administration 30 min before or 1 h after CLP on lung MIP-2 and RANTES levels in mice (n=6–9 in each group), which were divided into CLP-operated and sham-operated groups. CLP-operated mice received vehicle (DMSO in PBS, 0.25% v/v) or SR140333 (1 mg/kg; 0.25 mg/ml) s.c. 30 min before (pretreatment) or 1 h after (post-treatment) the CLP. The same surgical procedure as the CLP-operated animals, except CLP was performed on sham-operated animals. Eight hours after the CLP procedure, mice were killed, and lung MIP-2 and RANTES levels were determined as described in Materials and Methods. Results shown are the mean ± SEM. *, P < 0.001, when vehicle-treated, CLP animals were compared with sham group animals; **, P < 0.001, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals; {infty}, P < 0.05, when vehicle-treated, CLP animals were compared with sham group animals; {dagger}, P < 0.05, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals.

 
Similarly, MCP-1 production increased significantly in the lungs of CLP mice compared with that of sham animals (Fig. 4A ). This increase in MCP-1 levels was reversed significantly by SR140333 administered pre- or post-CLP surgery (Fig. 4A) . A similar trend was also found in plasma samples (Fig. 4B) . The high level of plasma MCP-1 found in CLP mice was decreased by SR140333 treatment. The reduction observed was especially significant when SR140333 was injected 30 min before CLP (Fig. 4B) . Moreover, there was a trend to reduce the plasma levels of MCP-1 when SR140333 was injected 1 h after CLP.


Figure 4
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  		Figure 4. Effect of SR140333 administration 30 min before or 1 h after CLP on lung and plasma MCP-1 levels in mice (n=6–9 in each group), which were divided into CLP-operated and sham-operated groups. CLP-operated mice received vehicle (DMSO in PBS, 0.25% v/v) or SR140333 (1 mg/kg; 0.25 mg/ml) s.c. 30 min before (pretreatment) or 1 h after (post-treatment) the CLP. The same surgical procedure as the CLP-operated animals, except CLP was performed on sham-operated animals. Eight hours after the CLP procedure, mice were killed, and plasma and lung MCP-1 levels were estimated as described in Materials and Methods. Results shown are the mean ± SEM. *, P < 0.01, when vehicle-treated, CLP animals were compared with sham group animals; **, P < 0.001, when vehicle-treated, CLP animals were compared with sham group animals; {infty}, P < 0.01, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals; {dagger}, P < 0.05, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals.

 
Effect of SR140333 treatment on cytokine levels in lung
As a primary line of defense against invading pathogens, cytokines are released in large amount by the host immune system. In our CLP sepsis model, we measured the major cytokines, IL-1ß, IL-6, and TNF-{alpha}, in lung tissue. As shown in Figure 5A , CLP animals injected only with the vehicle showed a significant increase in lung IL-1ß levels compared with that in sham mice. Administration of SR140333, 30 min before and 1 h after CLP, resulted in a significant reduction in the lung IL-1ß levels. Another important cytokine studied, IL-6, showed a similar pattern of increase in CLP-induced sepsis (Fig. 5B) . The lung levels of IL-6 in CLP mice injected only with vehicle, 30 min before or 1 h after CLP, were significantly higher compared with that in the sham-operated group. SR140333, when injected 30 min before or 1 h after CLP, decreased the lung IL-6 levels significantly compared with the corresponding values in the absence of SR140333 treatment in CLP mice (Fig. 5B) . The lung TNF-{alpha} level was not significantly different between sham-operated animals and CLP-operated mice treated only with the vehicle (Fig. 5C) . Further, treatment with SR140333 did not lower the lung TNF-{alpha} level significantly in both of the treatment groups 8 h after CLP, as shown in Figure 5C .


Figure 5
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  		Figure 5. Effect of SR140333 administration, 30 min before or 1 h after CLP, on lung levels of proinflammatory cytokines, IL-1ß, IL-6, and TNF-{alpha}, in mice (n=6–9 in each group), which were divided into CLP-operated and sham-operated groups. CLP-operated mice received vehicle (DMSO in PBS, 0.25% v/v) or SR140333 (1 mg/kg; 0.25 mg/ml) s.c. 30 min before (pretreatment) or 1 h after (post-treatment) the CLP. The same surgical procedure as the CLP-operated animals, except CLP was performed on sham-operated animals. Eight hours after the CLP procedure, mice were killed, and lung IL-1ß, IL-6, and TNF-{alpha} levels were estimated as described in Materials and Methods. Results shown are the mean ± SEM. *, P < 0.001, when vehicle-treated, CLP animals were compared with the sham group animals; **, P < 0.001, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals; {infty}, P < 0.01, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals; {dagger}, P < 0.05, when vehicle-treated, CLP animals were compared with sham group animals.

 
Effect of SR140333 treatment on adhesion molecules in the lung
Adhesion molecules play an important role in the leukocyte-endothelial interactions and resulting leukocyte migration into the site of injury or infection. ELISA assay was performed to analyze the lung levels of adhesion molecules such as ICAM-1, VCAM-1, and E- and P-selectin in sepsis. Figure 6A represents ICAM-1 levels in the lung of sham-operated or CLP-operated mice. Animals with CLP surgery had a significantly higher level of ICAM-1 in the lung compared with the level in the sham surgery group (Fig. 6A) . SR1403333 injected 30 min before or 1 h after CLP lowered this increase significantly (Fig. 6A) . The lung VCAM-1 level showed no statistically significant increase with CLP operation compared with the levels in sham-operated mice (Fig. 6B) .


Figure 6
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  		Figure 6. Effect of SR140333 administration 30 min before or 1 h after CLP on lung levels of adhesion molecules, ICAM-1 and VCAM-1, in mice (n=6–9 in each group), which were divided into CLP-operated and sham-operated groups. CLP-operated mice received vehicle (DMSO in PBS, 0.25% v/v) or SR140333 (1 mg/kg; 0.25 mg/ml) s.c. 30 min before (pretreatment) or 1 h after (post-treatment) the CLP. The same surgical procedure as CLP-operated animals, except the CLP was performed on sham-operated animals. Eight hours after the CLP procedure, mice were killed, and lung ICAM-1 and VCAM-1 levels were estimated as described in Materials and Methods. Results shown are the mean ± SEM. *, P < 0.05, when vehicle-treated, CLP animals were compared with sham group animals; **, P < 0.05, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals; {infty}, P < 0.01, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals.

 
Selectins are a major class of adhesion molecules known to play an important role in early inflammation stages in recruiting the leukocytes to the site of inflammation. Next, we investigated the changes in the production of selectins in lung, and the lung E-selectin level was significantly higher after CLP surgery compared with that in sham-operated mice (Fig. 7A ). This increase was reversed almost completely by SR1403333 when injected 30 min or 1 h after CLP (Fig. 7B) . A similar trend was observed in case of another selectin, the P-selectin. When P-selectin levels were determined 8 h after CLP procedure, there was a significant increase in pre- and post-vehicle control groups compared with sham-operated mice. Administration of SR140333 30 min before or 1 h after CLP resulted in a significant reduction in lung P-selectin levels. These results indicate that treatment with SR140333 reduced the lung inflammation in CLP sepsis in terms of neutrophil infiltration, levels of chemokines, cytokines, and adhesion molecules.


Figure 7
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  		Figure 7. Effect of SR140333 administration 30 min before or 1 h after CLP on lung levels of adhesion molecules, E-selectin and P-selectin, in mice (n=6–9 in each group), which were divided into CLP-operated and sham-operated groups. CLP-operated mice received vehicle (DMSO in PBS, 0.25% v/v) or SR140333 (1 mg/kg; 0.25 mg/ml) s.c. 30 min before (pretreatment) or 1 h after (post-treatment) the CLP. The same surgical procedure as CLP-operated animals, except the CLP was performed on sham-operated animals. Eight hours after the CLP procedure, mice were killed, and lung E-selectin and P-selectin levels were estimated as described in Materials and Methods. Results shown are the mean ± SEM. *, P < 0.001, when vehicle-treated, CLP animals were compared with sham group animals; **, P < 0.01, when vehicle-treated, CLP animals were compared with sham group animals; {infty}, P < 0.01, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals; {dagger}, P < 0.05, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals; {ddagger}, P < 0.05, when vehicle-treated, CLP animals were compared with sham group animals; #, P < 0.05, when SR140333-treated, CLP animals were compared with vehicle-treated, CLP animals.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Accumulating evidence over the years has emphasized the proinflammatory role of SP in various inflammatory states. SP, acting via NK-1 receptors, has been shown to enhance inflammation. SP is also known to activate mast cells and thus, stimulate neutrophil extravasations [28 , 29 ]. The importance of SP and NK-1 receptors in inflammation has been evident from the earlier studies using mice deficient in NK-1 receptors [26 ] and the PPT-A gene [10 ], which demonstrated a key role of SP in acute pancreatitis and associated lung injury. In addition, treatment with NK-1 receptor antagonist CP96345 has been shown to be protective against acute pancreatitis and associated lung injury [30 , 31 ]. Recently, we have reported the role of PPT-A gene products as key mediators of lung injury in polymicrobial sepsis [15 ]. The study showed that the deletion of the PPT-A gene in mice delayed the pathology of sepsis with protection against pulmonary tissue damage [15 ]. Results from the present study further substantiate the role of SP in sepsis and demonstrate SP as a potential therapeutic target in sepsis.

SR140333 is a highly selective and potent, nonpeptide antagonist of the NK-1 receptor compared with CP96345 and RP67580, two prototypical, nonpeptide antagonists of the NK-1 receptor [16 ]. This competitive blocker has the advantage of being species-independent in its potency [16 ]. Thus, we used SR140333 at a dose sufficient enough to inhibit the SP effects of bronchoconstriction and plasma extravasations [16 ]. CLP was used as the model of polymicrobial sepsis, and SR140333 was injected 30 min before or 1 h after the CLP surgery. The experiment was designed to study the effects of inhibition when SR140333 was given before the surgery as well as after the pathogenic assault has set in. The animals were killed 8 h after CLP to collect blood and tissue, as we observed the MPO activity to peak at this time-point (unpublished data). As the lung is the main target for damage in sepsis, we have focused primarily on pulmonary injury and the levels of inflammatory mediators in the lung.

Neutrophil migration to the site of infection is important in the control of infection in sepsis. MPO activity in the lung as a measure of neutrophil infiltration was evaluated 8 h after CLP. MPO activity increased after CLP and was reduced significantly by treatment with SR140333, 30 min before or 1 h after CLP. This was supported further by the histological sections of the lung. SR140333 injection clearly reduced the leukocyte infiltration and edema, the signs of lung injury in sepsis. To evaluate the consistent response of the NK-1 receptor blockade, a second NK-1 receptor antagonist, L703606, was administered 30 min before CLP. L703606 reduced the lung MPO activity significantly compared with saline-treated mice. Thus, a similar response was observed with both of the antagonists in terms of MPO levels.

Recruitment of various inflammatory cells including neutrophils is mediated by chemokines [32 ], MCP-1 and MIP-2, which are known to orchestrate migration of leukocytes during sepsis and lead to tissue injury. We have shown that MCP-1 and MIP-2 level in lung correlates with neutrophil infiltration in the lung [15 ]. Consistent with the earlier reports, we found a significant increase in the production of MCP-1 and MIP-2 in the lung. SR140333 treatment significantly lowered the lung levels of these two chemokines. RANTES levels were also reduced significantly with SR140333 administration.

Proinflammatory cytokines such as IL-1ß and TNF-{alpha} are needed to control infection in sepsis [4 ]. Although these cytokines recruit and activate cells that defend against pathogens during the early phase of infection, if produced in excess, the same cytokines can damage the tissue [3 ]. Further, MCP-1 is known to attract neutrophils by activating resident macrophages, which are the source of many inflammatory cytokines and chemokines [33 ]. Therefore, next, we studied the production of major cytokines IL-6, IL-1ß, and TNF-{alpha} in the lung after SR140333 treatment. As the cytokines produced locally in tissue inflammation are more important than that in the serum, we analyzed the levels mainly in lung tissues.

There was a significant increase in the lung levels of IL-6 and IL-1ß 8 h after CLP. Neuropeptides are known to stimulate cytokine production in macrophages, lymphocytes, and mast cells [34 ]. In addition, SP is reported to influence LPS-induced production of proinflammatory cytokines, which was abolished by NK-1 receptor blocking [34 ]. SR140333 administration 30 min before or 1 h after CLP in our study reversed the increase in IL-6 and IL-1ß. However, unlike in LPS-induced endotoxemia, in the CLP model of sepsis, TNF-{alpha} is not the main mediator of mortality [35 , 36 ]. Consistently, our results showed no significant difference in lung TNF-{alpha} level after CLP.

Adhesion molecules are important in the activation and adhesion of leukocytes to the endothelium and infiltration into the tissue to fight the infectious organisms [37 ]. Selectins, a major group of adhesion molecules, are involved in the earliest step of the acute inflammatory process mediating the rolling of leukocytes [37 ]. A high level of proinflammatory mediators in sepsis is reported to up-regulate various adhesion molecules [38 ]. Absence of ICAM-1 in knockout mice has been reported to reduce the severity of sepsis by impairing the leukocyte migration and damage of organs [39 ]. We found a significant increase in the lung levels of ICAM-1 and E- and P-selectin in mice with CLP-induced sepsis compared with the sham-operated group. Treatment with SR140333 lowered the lung levels of ICAM-1 and E- and P-selectin significantly. It has been shown that SP induces leukocyte trafficking via the up-regulation of adhesion molecules, and treatment with SR140333 reduced the leukocyte rolling, adhesion, and emigration [40 ]. Thus, SR140333 treatment in our study could have reduced the severity of sepsis by impairing the leukocyte migration via modulating the levels of adhesion molecules. Further, there was a significant reduction in the VCAM-1 level with SR140333 treatment in CLP mice, which is consistent with the reported blocking of SP-induced endothelial VCAM-1 expression in skin cells by a NK-1 receptor antagonist [41 ].

As SP levels are known to be increased in sepsis, it can be speculated from the present data that SP acting through NK-1 receptors is one of the major players in sepsis, responsible for the leukocyte responses, inflammatory processes, and pulmonary damage. We hypothesize further that chemokines (MCP-1, MIP-2), cytokines (IL-1ß, IL-6), and adhesion molecules (ICAM-1, E-selectin, and P-selectin) are modulated downstream by the action of SP on NK-1 receptors. Thus, blocking the NK-1 receptor by SR140333 could ameliorate the inflammatory effects in sepsis.

In summary, data from the present study show a beneficial role of SR140333 treatment in lung injury in the CLP-induced mouse sepsis model. SR140333 injected 30 min before or 1 h after CLP significantly reduced the lung levels of MPO, MIP-2, MCP-1, IL-1ß, IL-6, ICAM-1, E-selectin, and P-selectin. As septic lung injury involves various mediators, therapeutic strategies should be targeted at multiple mediators for a successive outcome, and NK-1 receptor blocking has a potential therapeutic benefit by lowering the leukocyte infiltration and lung levels of chemokines, cytokines, and adhesion molecules. However, further clinical studies are needed to establish the benefits of the NK-1 receptor blockade.


   ACKNOWLEDGEMENTS
 
This work was supported by a research grant from National Medical Research Council (grant no. R-184-000-111-213). We thank Sanofi Synthelabo (France) for providing SR140333 for the study.

Received April 10, 2007; revised May 8, 2007; accepted May 21, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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