Published online before print November 12, 2007
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Cardiovascular Biology Research Group, Department of Pharmacology, National University of Singapore, Singapore
1 Correspondence: Cardiovascular Biology Group, Department of Pharmacology c/o Centre for Life Sciences, #03-02, National University of Singapore, 28 Medical Drive, Singapore 117456. E-mail: mbhatia{at}nus.edu.sg
ABSTRACT
Endotoxemia is a life-threatening, inflammatory condition that involves multiple organ injury and dysfunction. Preprotachykinin-A (PPT-A) gene products, substance P (SP), and neurokinin-A have been shown to play an important role in neurogenic inflammation. To investigate the role of PPT-A gene products on multiple organ injury in LPS-induced endotoxemia, endotoxemia was induced by LPS administration (10 mg/kg, i.p.) in PPT-A gene-deficient mice (PPTA–/–) and the wild-type (WT) control mice (PPT-A+/+). I.p. administration of LPS to WT mice caused a significant increase in circulating levels of SP as well as in liver, lung, and kidney. PPT-A gene deletion significantly protected against liver, pulmonary, and renal injury following LPS-induced endotoxemia, as evidenced by tissue myeloperoxidase activities, plasma alanine aminotransferase, aspartate aminotransferase levels, and histological examination. Furthermore, PPT-A–/– mice had significantly attenuated chemokines, proinflammatory cytokines, and adhesion molecule levels in the liver, lung, and kidney. These results show that PPT-A gene products are critical proinflammatory mediators in endotoxemia and the associated multiple organ injury. In addition, the data suggest that deletion of the PPT-A gene protected mice against organ damage in endotoxemia by disruption in neutrophil recruitment.
Key Words: substance P • endotoxin shock • neutrophils • inflammation
INTRODUCTION
Endotoxemia is marked by activation of inflammatory responses, which can lead to shock, multiple organ failure, and the suppression of immune and wound-healing processes. Severe sepsis and septic shock constitute one of the leading causes of mortality among Intensive Care Unit and Post-Operational Care patients [1 ]. Endotoxemia is a condition in which endotoxin, a component of the outer wall of gram-negative microorganisms, accesses the blood stream, disseminates through the body, and initiates production of biologically active molecules from a variety of cells, including cytokines, bioactive amines, eicosanoids, and a variety of reactive oxygen species [2 ]. These mediators alone or through their interaction recruit neutrophils to accumulate, leading to local inflammation and further, to septic shock [3 ]. Sequestration of neutrophils in a variety of vascular beds is regarded as the critical and first step to induce leukocyte-related vascular injury in endotoxemia. Neutrophils have a pivotal role in the defense against bacterial infections, as shown by neutropenia, which increases susceptibility to infection and to sepsis. However, overwhelming activation of neutrophils is known to elicit tissue damage.
Substance P (SP) and neurokinin A (NKA), neuropeptide products of the preprotachykinin-A (PPT-A) gene, have been shown to play an important role in neurogenic inflammation. Hallmarks of neurogenic inflammation are increases in vascular permeability, plasma extravasation, edema formation, and leukocyte infiltration [4 5 ]. SP and NKA preferentially bind to the G protein-coupled NK-1R and NK-2R, respectively [6 ]. SP enhances cytokine secretion from lymphocytes, monocytes, macrophages, and mast cells [7 8 9 10 11 12 13 14 15 ]. Furthermore, SP-induced release of inflammatory mediators such as cytokines and histamine potentiates tissue injury and further stimulates leukocyte recruitment, thereby amplifying the inflammatory response [16 ]. SP also elicits local vasodilatation and alters vascular permeability, thus enhancing the delivery and accumulation of leukocytes to tissues for the expression of local immune responses [17 ]. SP can specifically stimulate the chemotaxis of neutrophils [18 19 20 ]. By promoting vasodilatation, leukocyte chemotaxis, and leukocyte/endothelial cell adhesion, SP ensures the extravasation, migration, and subsequent accumulation of leukocytes at sites of injury [21 ].
We have shown earlier that mice deficient in the PPT-A gene (PPT-A–/–) are protected against acute pancreatitis [22 ] and cecal ligation and puncture (CLP)-induced sepsis [23 ]. Other investigators also reported the role of the PPT-A gene in airway inflammation [24 25 ], arthritis [26 ], cystitis, and inflammatory bowel disease [27 ]. These results show that PPT-A gene products are critical inflammatory mediators. Moreover, pretreatment of mice with NK-1R antagonists, CP-96,345 and L-733,060, dose-dependently protected mice from galactosamin (GalN)/LPS-induced liver injury [28 ]. Studies using NK-1R antagonists or mice genetically deficient in the NK-1R have proven a role for this receptor in asthma and chronic bronchitis, intestinal inflammation, pancreatitis [29 30 31 ], and resistance to infection [4 32 ]. However, NK-1R is the binding site for other ligands in addition to SP, and not all are products of PPT-A gene. Hence, in this study, we investigated the effects of genetically deleting PPT-A on the pathogenesis of LPS-induced endotoxemia and associated liver, pulmonary, and renal injury in mice. We have also identified the mechanisms by which proinflammatory mediator SP contributes to tissue damage during endotoxemia.
MATERIALS AND METHODS
Induction of LPS-challenged sepsis
All animal experiments were approved by the Animal Ethics Committee of National University of Singapore and were performed in accordance with established International Guiding Principles for Animal Research. PPT-A–/– mice were a gift from Professor Allan Basbaum (University of California, San Francisco, CA, USA) and bred as described previously [33
]. Male BALB/c (25–30 g) were maintained in the Animal Housing Unit of this university in an environment with controlled temperature (21–24°C) and lighting (12:12 h light-darkness cycle). Standard laboratory chow and drinking water were provided ad libitum. A period of 2 days was allowed for animals to acclimatize before any experimental manipulations were undertaken. LPS-challenged endotoxemia was induced as described previously [34
]. Mice were randomly assigned to control or experimental groups using 10 or more animals for each group. Normal saline or saline-containing bacterial endotoxin polysaccharide (LPS, Escherichia coli, serotype O127:B8, Sigma-Aldrich, Germany; 10 mg/kg, i.p.) was administered to mice. Six hours after the LPS injection, mice were killed by an i.p. injection of a lethal dose of 50 mg/kg pentobarbital (Nembutal, CEVA Sante Animale, Naaldwijk, Netherlands). Liver, lung, and kidney tissues were removed and stored at –80°C for biochemical and histological assays as described below.
Measurement of SP
Samples of liver, lung, kidney, and plasma were collected from the animals. The tissue fragments were homogenized using Heidolph Diax 900 (Schwabach, Germany) in 2 ml ice-cold assay buffer for 20 s. The homogenates were centrifuged (13,000 rpm, 20 min, 4°C), and the supernatants were collected. The supernatant was adsorbed on Sep-Pak C18 cartridge columns (Waters Associates, Milford, MA, USA), as described elsewhere [30
]. The adsorbed peptide was eluted with 1.5 ml 75% v/v acetonitrile. The samples were freeze-dried and reconstituted in sample buffer. SP content was then determined with an ELISA kit (Peninsula Laboratories, San Carlos, CA, USA), according to the manufacturer’s instructions. The absorbance was measured at 450 nm by a microplate reader (SPECTRAFluor Plus, Tecan Austria GmbH, Grödig, Austria) and was then corrected for the DNA content of the tissue samples. DNA assay was performed fluorometrically by using Hoechst dye 33256 by the method of Labarca and Paigen [35
] and salmon testes DNA as standard. SP can be measured in the range of 0–10 ng/ml in this assay. SP concentration is expressed as ng/ml for plasma and ng/µg DNA for tissue.
Myeloperoxidase (MPO) estimation
Neutrophil sequestration in lung, liver, and kidney was quantified by measuring tissue MPO activity. 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-Aldrich). The suspension was subjected to four cycles of freezing and thawing and was distrupted further 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 (TMB) substrate (KPL, Gaithersburg, MD, USA), 80 mM sodium phosphate buffer (pH 5.4), and 0.3 mM hydrogen peroxide. This mixture was incubated at 37°C for 2 min, the reaction was terminated with 2 N H2SO4, and the absorbance was measured spectrophotometrically at 450 nm. The absorbance was then corrected for the DNA content of the tissue samples and expressed as the fold-increase over control. DNA assay was carried out as described earlier.
Examination of liver injury
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using a kinetic spectrophotometric assay (Infinity, Thermo Electron Corp., Grenoble, France), according to the manufacturer’s instructions. AST and ALT were measured by the decrease in absorbance at 340 nm, which is directly proportional to the oxidation of NADH to NAD. The activities of ALT and AST were expressed as an international unit (U/L).
ELISA analysis
For the measurement of cytokines (IL-1β, IL-6, and TNF-
), chemokines (MCP-1 and MIP-1), and adhesion molecules (VCAM-1, ICAM-1, P-selectin, and E-selectin) in homogenized liver, lung, and kidney, ELISA kits from R&D Systems (Minneapolis, MN, USA) were used according to the manufacturer’s instructions. The lower limits of detection of the levels of MIP-2, MIP-1, IL-1β, TNF-, IL-6, VCAM-1, ICAM-1, P-selectin, and E-selectin were 15.625, 7.8125, 15.625, 15.625, 31.25, 62.5, 62.5, 31.25, and 31.25 pg/ml, respectively. Briefly, primary antibody was aliquoted onto ELISA plates and incubated at 4°C overnight. Samples and standards were incubated for 2 h, the plates were washed, and a biotinylated secondary antibody was added for 2 h. Plates were washed again, and streptavidin bound to HRP was added for 20 min. Following a further wash, TMB was added for color development, and the reaction was terminated with 2 N H2SO4 after 20 min incubation. The absorbance was measured at 450 nm using spectrophotometry and was then corrected for the DNA content of the tissue samples. DNA assay was carried out as described earlier. Results were expressed as pg/µg DNA.
Morphological examination
Lung, liver, and kidney segments were fixed in 10% v/v phosphate-buffered formalin for 24 h and then embedded in paraffin. Next, the samples were sectioned (5 µm) using a microtome, stained with H&E, and examined with light microscopy at x400 magnifications.
Statistical analysis
Data are expressed as the means ± SEM. The statistical significance was evaluated by one-way ANOVA, followed by post-hoc Tukey’s test for multiple comparisons. A P value of <0.05 was considered to indicate a significant difference. In all figures, vertical error bars denote the SEM. The absence of such error bars indicates that the SEM falls within the dimensions of the data point.
RESULTS
Effects of LPS-induced endotoxemia on SP levels
SP levels were increased significantly 6 h after LPS administration in plasma (Fig. 1A
), liver, lung, and kidney (Fig. 1B)
in the wild-type (WT) mice compared with control. No SP was detected in PPT-A–/– mice (data not shown).
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Figure 1. Plasma and tissue SP levels in WT mice subjected to LPS-induced endotoxemia. WT mice (n=10 in each group) were induced by a single injection of LPS (10 mg/kg, i.p.) or saline. Six hours after the LPS injection, mice were killed, and plasma SP levels (A) and SP levels in liver, lung, and kidney (B) were measured as described in Materials and Methods. Results shown are the means ± SEM. *, P < 0.05, when LPS-treated animals were compared with saline-treated animals.
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Figure 2. Effect of PPT-A gene deletion on LPS-induced MPO activities. WT and PPT-A–/– mice (n=10 in each group) were induced by a single injection of LPS (10 mg/kg, i.p.) or saline. Six hours after the LPS injection, mice were killed, and liver, lung, and kidney MPO activities were measured as described in Materials and Methods. Results shown are the means ± SEM. *, P < 0.05, when LPS-treated animals were compared with saline-treated animals.  , P < 0.05, when LPS-treated WT were compared with LPS-treated PPT-A–/–.
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Figure 3. Effect of PPT-A gene deletion on LPS-induced liver injury. WT and PPT-A–/– mice (n=10 in each group) were induced by a single injection of LPS (10 mg/kg, i.p.) or saline. Six hours after the LPS injection, mice were killed, and plasma ALT levels (A) and AST levels (B) were measured as described in Materials and Methods. Results shown are the means ± SEM. *, P < 0.05, when LPS-treated animals were compared with saline-treated animals. , P < 0.05, when LPS-treated WT were compared with LPS-treated PPT-A–/–.
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levels (Fig. 4A
and 4B
). PPT-A gene deletion in mice caused a significant reduction in MIP-2 and MIP-1 levels in the liver. MIP-2 and MIP-1 levels were also elevated in lung and kidney of LPS-challenged mice compared with control, and the elevations were reduced significantly in LPS-induced PPT-A knockout mice.
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Figure 4. Effect of PPT-A gene deletion on tissue chemokine levels following LPS-induced endotoxemia. WT and PPT-A–/– mice (n=10 in each group) were induced by a single injection of LPS (10 mg/kg, i.p.) or saline. Six hours after the LPS injection, mice were killed, and MIP-2 (A) and MIP-1 (B) levels in liver, lung, and kidney were measured as described in Materials and Methods. Results shown are the means ± SEM. *, P < 0.05, when LPS-treated animals were compared with saline-treated animals. , P < 0.05, when LPS-treated WT were compared with LPS-treated PPT-A–/–.
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, and IL-6 in liver, lung, and kidney (Fig. 5A
5B
5C
). However, PPT-A gene deletion resulted in a significant reduction in the protein levels of IL-1β, TNF-, and IL-6 in liver, lung, and kidney 6 h after the induction of endotoxemia.
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Figure 5. Effect of PPT-A gene deletion on tissue cytokine levels following LPS-induced endotoxemia. WT and PPT-A–/– mice (n=10 in each group) were induced by a single injection of LPS (10 mg/kg, i.p.) or saline. Six hours after the LPS injection, mice were killed, and levels of IL-1β (A), TNF- (B), and IL-6 (C) in liver, lung, and kidney were measured as described in Materials and Methods. Results shown are the means ± SEM. *, P < 0.05, when LPS-treated animals were compared with saline-treated animals. , P < 0.05, when LPS-treated WT were compared with LPS-treated PPT-A–/–.
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Figure 6. Effect of PPT-A gene deletion on tissue adhesion molecule levels following LPS-induced endotoxemia. WT and PPT-A–/– mice (n=10 in each group) were induced by a single injection of LPS (10 mg/kg, i.p.) or saline. Six hours after the LPS injection, mice were killed, and levels of VCAM-1 (A), ICAM-1 (B), P-selection (C), and E-selectin (D) in liver, lung, and kidney were measured as described in Materials and Methods. Results shown are the means ± SEM. *, P < 0.05, when LPS-treated animals were compared with saline-treated animals. , P < 0.05, when LPS-treated WT were compared with LPS-treated PPT-A–/–.
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Figure 7. Morphological changes in mouse liver sections of WT and PPT-A–/– on LPS induction of endotoxemia (H&E, original magnification, x40). (A) Control, WT. (B) Control, PPT-A–/–. (C) LPS-induced endotoxemia in WT mice. (D) LPS-induced endotoxemia in PPT-A–/– mice.
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Figure 8. Morphological changes in mouse lung sections of WT and PPT-A–/– on LPS induction of endotoxemia (H&E, original magnification, x40). (A) Control, WT. (B) Control, PPT-A–/–. (C) LPS-induced endotoxemia in WT mice. (D) LPS-induced endotoxemia in PPT-A–/– mice.
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Figure 9. Morphological changes in mouse kidney sections of WT and PPT-A–/– on LPS induction of endotoxemia (H&E, original magnification, x40). (A) Control, WT. (B) Control, PPT-A–/–. (C) LPS-induced endotoxemia in WT mice. (D) LPS-induced endotoxemia in PPT-A–/– mice.
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In WT mice, endotoxemia was induced by injecting a stimulating dose of LPS (10 mg/kg) for 6 h to study early LPS inflammatory responses. We have shown that 6 h after LPS administration, plasma and tissue (liver, lung, and kidney) SP levels were elevated significantly compared with control animals, indicating that SP released after the onset of endotoxemia. Notably, the increase in tissue SP levels during endotoxemia was greater than that in plasma SP level, suggesting that intrinsic neurons to the airways, liver, or kidney release more SP into local tissues than into the circulation and that SP may preferentially act in a paracrine or autocrine manner in LPS-induced endotoxemia. Furthermore, our earlier results have demonstrated that plasma and lung levels of SP were also increased in CLP-induced sepsis [23 ], and SP levels in plasma, pancreas, and lungs were found to be elevated in caerulein-induced acute pancreatitis [29 ]. Moreover, another report has shown LPS caused significant increases in lung PPT-A mRNA expression, bronchoalveolar lavage (BAL) SP, and calcitonin gene-related peptide (CGRP) levels [36 ]. SP is released from capsaicin-sensitive C fibers, which are a subpopulation of sensory neurons containing SP, NKA, CGRP, and transient receptor potential vanilloid (TrpV)-1. It has also been shown that capsaicin-sensitive C fibers coexpress TLR4 and CD14, which function as pattern recognition receptors for LPS [37 ], suggesting direct stimulation of nociceptors and release of neuropeptides by LPS. Studies have shown that fever production in response to LPS was significantly attenuated in TrpV1–/– mice [38 ] and rat pretreated with SP antagonist [39 ], indicating that neurogenic inflammation may play a detrimental role in LPS-induced endotoxemia.
LPS-induced endotoxemia was characterized by sequestration of neutrophils in the liver, lung, and kidney (increased tissue MPO activities) and increased ALT and AST, markers of liver injury, as well as morphological evidence of tissue injury. Our results show that deletion of PPT-A results in a marked decrease in all of the parameters that characterize the severity of LPS-induced endotoxemia. Our results in the present study demonstrate that PPT-A gene-encoded NKs play a key role in the pathogenesis of LPS-induced tissue injury in endotoxemia. Likewise, pretreatment of mice with antagonists of the SP-specific NK-1R dose-dependently protected mice from GalN/LPS-induced liver injury, and NK-1R blockade reduced inflammatory liver damage, i.e., edema formation, neutrophil infiltration, hepatocyte apoptosis, and necrosis [28 ]. Furthermore, we have shown that PPT-A gene deletion protected mice against CLP-induced sepsis [23 ] and caerulein-induced acute pancreatitis [22 ]. These findings provide further support for the concept that tachykinins act as potential therapeutic targets in inflammation [40 ].
In the present study, the PPT-A–/– mice showed significantly lower MIP-2 levels in liver, lung, and kidney homogenates than WT mice during endotoxemia. The same trend was observed in MIP-1 levels in liver, lung, and kidney homogenates of PPT-A–/– mice. In a CLP-operated sepsis model, plasma and lung MIP-2 levels were shown to be up-regulated, and deletion of the PPT-A gene reduced the plasma and lung MIP-2 levels significantly [23
]. Our previous work illustrated that SP primed neutrophils for chemotactic responses to the CXC chemokine, MIP-2, and the CC chemokine, MIP-1 [41
]. In addition, blockade of NK-1R attenuates CC and CXC chemokine productions in experimental acute pancreatitis and associated lung injury [42
]. MIP-2 has been shown to play a significant role in a LPS-induced, inflammatory response in rat lungs and is required for the full recruitment of neutrophils [43
]. Pretreatment with mAb against MIP-2 abolished extravascular recruitment of polymorphonuclear (PMN) leukocytes in the livers of endotoxemic mice [44
]. On the other hand, anti-MIP-1 administered at LPS-induced injury resulted in significant reductions in BAL neutrophils as well as in injury as measured by pulmonary vascular permeability [45
]. These data provide evidence that the mechanism by which PPT-A gene products play a crucial role in LPS-induced tissue damage is by modulating PMN leukocyte recruitment through CXC and CC chemokines, such as MIP-2 and MIP-1.
We have also shown that there are marked reductions in levels of proinflammatory cytokine IL-1β, TNF-, and IL-6 in liver, lung, and kidney homogenates in PPT-A–/– mice compared with normal mice during endotoxemia. In another report, liver injury-induced lung inflammation was shown to be mediated predominantly by IL-1β, and knockdown of IL-1β expression, prior to hepatic injury, led to significant reductions in cytokine production and pulmonary neutrophil accumulation [46
]. Studies have revealed production of TNF-, IL-1β, and IL-6 by rat PMN leukocyte subpopulations after exposure to SP. LPS enhanced SP-mediated neutrophil adherence and associated IL-1β and TNF- release partly through NK-1R [47
]. MIP-2 and MIP-1, possibly released by TNF- stimulation of macrophages, are associated with acute lung injury, possibly by inducing neutrophil chemotaxis [48
], and up-regulate vascular adhesion molecules required for neutrophil influx [45
], respectively. TNF-, IL-1β, and MIP-1 have been shown to play an essential role in the LPS-induced acute lung injury [49
50
]. Thus, the current study shows that SP increases proinflammatory cytokine IL-1β, TNF-, and IL-6 levels and potentially, further alters the chemokine levels.
PPT-A gene deletion effectively reduced the protein expression of VCAM-1, ICAM-1, P-selectin, and E-selectin in the liver of LPS-induced endotoxemia. In the lung, VCAM-1, and P- and E-selectin protein levels were markedly reduced in PPT-A–/– endotoxemic mice compared with WT endotoxemic mice, whereas in the kidney, VCAM-1 and P-selectin were clearly decreased in PPT-A–/– endotoxemic mice. The results in the present study provide further evidence of the differential regulation of the adhesion molecules in liver, lung, and kidney in endotoxemia. Pretreatment with an anti-P-selectin antibody markedly reduced PMN leukocyte rolling and firm adhesion in endotoxemic mice [51
]. In acute pancreatitis, treatment with CP-96,345 effectively reduced the mRNA expression of P-selectin and E-selectin but not ICAM-1 and VCAM-1 in the pancreas and also suppressed E- selectin and P-selectin elevation in the lung. The highly regulated process of PMN leukocyte recruitment involves the participation of a wide range of adhesion proteins and signaling molecules. This has suggested that the protective effect of PPT-A gene deletion was mediated by the change in the regulation of a number of adhesion molecules, and ICAM-1, VCAM-1, E-selectin, and P-selectin are only a few of the major contributors of the recruitment process. In fact, SP has been shown to affect the levels of other adhesion molecules such as LFA-1 [52
53
], integrin -5 [54
], and complement receptor-associated OKM1 molecule [55
]. Taken together, our findings suggest that PPT-A gene deficiency could reduce the overproduction of proinflammatory cytokines and chemokines and suppress the widespread up-regulation of adhesion molecules in endotoxemia, thus assuaging the systemic inflammatory response and protecting mice against endotoxemia. However, in the present study, it is difficult to rule out the possibility that the protective effect of PPT-A gene deletion in endotoxemia may be attributed to delaying rather than alleviating the systemic inflammatory response.
In addition, it has to be noted that genetic depletion of SP could only partially reduce the tissue MPO activity and tissue levels of cytokines, chemokines, and adhesion molecules in LPS induced-endotoxemia. It suggests that other factors in addition to SP are involved in regulating inflammatory response in endotoxemia, which is clearly known to be a multifactorial process. For instance, LPS interacts directly with TLR4 and then initiates multiple intracellular signaling cascades, leading to the activation of NF-
B and subsequent transcription of genes for the proinflammatory mediators.
In conclusion, we have shown the mechanisms by which PPT-A gene products act as important proinflammatory mediators to regulate leukocyte recruitment and trafficking during LPS-induced inflammatory response, thus leading to multiple organ injury.
ACKNOWLEDGEMENTS
This work was supported by National Medical Research Council Research grant R-184-000-111-213. We thank Professor Allan Basbaum (University of California, San Francisco, CA, USA) for the gift of PPT-A–/– mice. We also thank Mei Leng Shoon (Department of Pharmacology, National University of Singapore) for her technical assistance and Yi Quan Tay, Ee Yong Loo, and Wai Mun Low (Animal Holding Unit, National University of Singapore) for their help.
Received August 27, 2007; revised October 9, 2007; accepted October 11, 2007.
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