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Substance P involves hepatic immune injury induced by concanavalin A in mice and stimulates cytokine synthesis in kupffer cells Abstract Objectives: To set up the mouse model of hepatic immune injury induced by concanavalin A (ConA) and investigate the role of substance P and neurogenic inflammation mediated by SP in this hepatitis model. we also cultured Kupffer cells to examine the functional consequences of exposing mouse Kupffer cells to SP and to determine whether it leads to proinflammatory signaling, such as production of cytokines. Methods: The mouse model of hepatic immune injury was induced by ConA, SP in liver homogenates was detected by enzyme-linktimmunosorbent assay (ELISA), Meanwhile, we also evaluated the protective effects of neurokinin receptor 1(NK1R)antagonist L-703,606 on hepatic immune injury with assessment of serological, histological. Kupffer cells from mice were isolated with NK1R expression evaluated by immunofluorescence and real-time PCR. Cytokine release was evaluated by ELISA. Results: The levels of sp were significantly increased in the model group compared with the control group(387.23 ± 29.36 vs 138.52 ±13.23pg/mL, P<0.05). The levels of serum ALT/AST were significantly increased in the model group and dramatically decreased in the L-703,606 treated group (782.37 ±21.51/1004.25 ± 18.24 U/L vs 402.22 ±16.42/581.45 ± 17.51 U/L,respectively;P<0.05). There were edema formation, hepatocellular apoptosis and granulocyte infiltration in livers of model group, which was significantly reduced by L-703,606. Mouse kupffer cells expressed NK1R and SP increased NK-1RmRNA expression in KC in vitro, while NK1R blockade abolished the effect of sp on NK1R mRNA expression and significantly reduced NK1R mRNA expression compared with the control culture medium. The cytokine levels (IL-6/TNF-α) in supernatant from the release of cultured kupffer cells revealed a substantial increase in the sp pretreated group as compared with the control culture medium group, (142.12 ± 11.09/72.15 ± 8.56 pg/ml vs 71.13 ± 9.36/31.02 ± 7.26 pg/ml, respectively; P< 0.05).The IL-6/TNF-α levels of supernatant in L-703,606 pretreated group were significantly lower than the sp pretreated group (142.12 ± 11.09/72.15 ± 8.56 pg/ml vs 68.16 ± 9.51/28.38 ± 5.04 pg/ml, P<0.05). Conclusion: Neurogenic inflammation induced by SP play an important role in hepatitis. Mouse kupffer cells express NK1R and SP increases NK1RmRNA expression in KC in vitro. SP enhances IL-6 and TNF-αsecretion and NK1R antagonist inhibits SP-Induced IL-6 and TNF-αrelease from KC in vitro. Key words: Concanavalin A; liver injury; substance P; neurokinin 1 receptor antagonist; neurogenic inflammation Inflammatory disease of the liver, i.e., hepatitis, represents a major problem in clinical medicine. Although hepatitis represents a heterogeneous group of diseases with different etiologies and complex pathogenesis, previous studies have indicated the immune system plays a pivotal role in most of these processes. The nervous and immune systems have been demonstrated to interact to modulate the immune response via secretion of neuropeptides. Neurogenic inflammation encompasses a series of inflammatory responses triggered by the activation of primary sensory neurons and the subsequent release of inflammatory neuropeptides[1].The neuropeptide substance P (SP) is an 11 amino acid peptide encoded by the preprotachykinin-A (PPT-A) gene. which is distributed throughout the nervous system of human and animal species[2]. It is a potent proinflammatory mediator and plays an important role in inflammation and viral infections[3].SP is a member of the tachykinin family of neuropeptides which interact with three natural tachykinin (neurokinin) receptors: NK‐1R, NK‐2R, and NK‐3R[4],but it effects are mainly mediated through the NK1R, a GPCR expressed in many tissues, including the nervous system, gastrointestinal tract, and cells of the immune system[3]. SP has been established to exert a vast range of proinflammatory effects in vitro and in vivo, influencing many immune and inflammatory disorders of the respiratory, gastrointestinal, and musculoskeletal systems. Although SP is a peptide of neuronal origin it is also found in non-neural cells including endothelial cells, macrophages, granulocytes, lymphocytes and dendritic cells. It stimulates immune cells to produce inflammatory cytokines, including interleukin (IL)-1, IL-6, tumour necrosis factor (TNF), interferon (IFN)-γ, and macrophage inflammatory protein 1β. SP induces chemotaxis and degranulation of neutrophils also stimulates respiratory burst[5]as well. Therefore, it is obvious that an extensive neuro-immune intersystem crosstalk exist between SP and the inflammatory response to injury.But limited data are available regarding the role of primary afferent neurons in the liver under both physiological and pathophysiological conditions. Studies have shown that primary afferent sensory neurons are necessary for disease activity in T cell-mediated immune hepatitis in mice[6].Renate Bane et al first demonstrated the presence of the NK-1R in mouse liver where it appeared predominantly in nonparenchymal mononuclear cells but also in hepatocytes.They identified that the NK-1R in NPC enriched in Kupffer cells, which are the cell population primarily activated by LPS in the liver[7].But in their study, there was neither detection of SP nor experiment in vitro. Therefore, in the present study, we investigated the effect of substance P in ConA-induced hepatitis model, we also cultured Kupffer cells to examine the functional consequences of exposing mouse Kupffer cells to SP and to determine whether it leads to proinflammatory signaling, such as production of cytokines. Methods. Drugs and materials ConA、L-703,606、Substance P、Collagenase IV、Triton X-100 were purchased from Sigma Chemical Company(St, Louis, Mo, USA), Percoll、RPMI 1640 medium and Fetal bovine serum (FBS) were purchased from Gibco Company(Gibco, USA),The neurokinin 1 receptor antibody NB300-101 and rabbit anti-NK-1R secondary antibody were purchased from Novus Biologicals (Novus,USA), IL-6 and TNF-αELISA kits was purchased from (R&D Systems, Inc. USA) Other commercial chemicals used in experiments were of analytical grade. Animal experiments and drug treatment Male Swiss albino mouse (25–30g), were purchased from the Experimental Animal Center of Shandong University School of Medicine, Shandong, PR China. The animals were housed 6 per cage under standardized conditions (25±30C, 12h light/dark cycle, humidity 50±10%) with free access to pelleted food and tap water. All experiments were conducted with the approval of the Institutional Experimental Animal Care and Use Committee of Shandong University. After acclimation for 6–7 days, animals were randomly divided into three groups as control group (group 1), ConA model group (group 2), NK1-receptor antagonist group (group 3), with each group containing 12 mice. Liver injury was induced as previously described[8]. ConA was dissolved in pyrogen-free phosphate-buffered saline(PBS),and intravenously injected (25 mg/kg) into group 2 mice via the tail vein. Mice in group 1 were injected with saline only. Mice in group 3 were administrated with NK1-receptor antagonist L-703,606 in dose of 10 mg/kg before Con A challenge. 6 hours later all mice were anesthetized to gain blood from eye sockets and then killed for liver. Measurement of Substance P Levels Liver samples were thawed, weighed, and homogenized at a ratio of 1:9 (w/v) in 0.9% saline solution. The homogenate was then centrifuged at 2000-3000 rpm for 10 min at 4℃.The levels of SP in the supernatant were measured using a mouse Substance P ELISA kit according to the manufacturer’s instructions. A 96-well microplate was loaded with 25ul of primary antibody specific for rat SP. 50ul of each sample and 50ul of the standard SP dilutions (as a control) mixed in the assigned wells in duplicate followed by the addition of biotinylated SP into each well except the Blank. The plate was incubated for 2h at room temperature, and then washed six times with provided in the kit wash buffer. Subsequently, 100ul of biotinylated anti-SP antibody solution was added, incubated for 1h, and then washed four times. 100ul of streptavidin-horseradish peroxidase conjugate solution was added to each well except chromogen blank, incubated for 1h, and washed again. After that, 100ul of substrate solution provided in the kit was added to each well and the plate was incubated for 1h at room temperature. The reaction was stopped with 2N HCl and the optical density values were read at 492 nm. The concentration of SP was expressed in pg/ml. Measuring serum liver enzymes Blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes. After centrifugation of whole blood (3000 r/min for 10 min at room temperature), serum was stored at -70℃ until analysis. The activities of alanine aminotransferase (ALT, a specific marker for hepatic parenchymal injury), and aspartate aminotransferase (AST, a nonspecific marker for hepatic injury) in serum were determined in units per liter using standard auto-analyzer methods on a Hitachi Automatic Analyzer (Hitachi, Inc, Japan). Histology The livers were removed, fixed with 4% phosphate-buffered paraformaldehyde and embedded in paraffin. Tissue sections (4um) were prepared and stained with hematoxylin/eosin; sections were then examined under light microscopy. In each section, three randomly selected areas were screened for edema, granulocytes, and hepatocyte apoptosis and necrosis. Isolation and culture of Kupffer cells Kupffer cells from mice were isolated by collagenase digestion and differential centrifugation, using Percoll density gradients as described previously[9]. The liver was perfused in vitro through the vena cava with 80ml Hanks’balanced salt solution (HBSS) Ca2+/Mg2+-free at 37℃, and transferred to a 100mm culture dish and perfusion was continued with complete HBSS containing 0.05% collagenase IV and 3mmol/L Ca2+ at 37℃. Liver tissue was finely diced into 2 mm3 sized pieces and the suspension incubated under constant agitation at 37 ℃ for 30 min. The liver homogenate was filtered through gauze mesh and the cells suspension was centrifuged at 50g for 3 min at 4 ℃ to remove hepatocytes. The non-parenchymal cells-enriched supernatant was centrifuged at 400g for 6 min. The cell pellet was resuspended in 30% Percoll with a density of 1.040 g/mL, and this was carefully layered onto 60% Percoll with a density of 1.075 g/mL. The double layer discontinuous gradient formed was overlaid with 3ml of HBSS and centrifuged at 400g for 15 min at 4℃. The opaque interface was collected, resuspended in HBSS and centrifuged at 400 g for 5 min at 4℃. The cells were seeded onto tissue culture plates at a density of 2×106/mL and cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 U/mL Penicillin/Streptomycin and 10 mmol/L HEPES at 37℃ with 5% CO2. All adherent cells phagocytosed latex beads and stained positive for catalase, confirming that they were Kupffer cells, and cells were cultured for 24 h before experiment. NK1R Immunofluorescence Cells (5×104) were placed on poly-L-lysine-coated slides in 200ul media, fixed with 4% paraformaldehyde, washed thrice in 0.1M PBS, blocked with serum-free protein, and permeabilized using 0.4% Triton X-100. Fixed cells were incubated overnight at 4℃ in the neurokinin 1 receptor antibody NB300-101 (1:50), followed by PBS washes and incubation with rabbit anti-NK-1R secondary antibody (1:500) for 1 h at room temperature. Then sections were washed in PBS and mounted with DAPI containing mounting medium (Vector Laboratories, Burlingame, CA; Nikon Inc., Melville, NY). Images were acquired by laser scanning confocal microscopy (LSM710; Carl Zeiss, Oberkochen, Germany) and analyzed by use of Image Pro Plus 6.0 (Media Cybernetics). Real-time PCR analysis Total RNA was extracted from KC using Tri-Reagent (Molecular Research Center, Cincinnati, OH) and quantitated and reverse-transcribed using the AffinityScript qPCR cDNA synthesis kit (Stratagene, Cedar Creek, TX, USA)according to the manufacturer's instructions. Total NK1R mRNA was quantified as described previously[10] using a primer set specific for a 109-bp fragment of NK1R transcripts (sense: 5′-GCATACACCGTAGTGGGAATC-3′;antisense:5′-CATCATTTTGACC ACCTTGC-3′). GAPDH was amplified as a control (sense: 5′-GGTGGTCTCCTCTGACTTCAACA-3′;antisense:5′-GTTGCTGTAGC CAAATTCGTTGT-3′).RT-PCR was conducted with cycling conditions consisting of 15 minutes Taq activation at 95℃ followed by denature, anneal, and extension phases for 15 seconds at 94 ℃, 30 seconds at 54℃, and 30 seconds at 72℃, respectively, for 40 cycles. The results for NK1R are expressed as the ratio to GAPDH. Measurement of cytokine release Micromolar concentrations of SP are usually used in vitro studies to identify their biological activities on immune cells and especially monocytes[11].Kupffer cells were seeded into 24-well plates at a density of 1×106/well and incubated with fresh DMEM containing 100ng/mL lipopolysaccharide(LPS) at 37 ℃ with 5% CO2. When KC were cultured without and with SP, or a combination of SP and L703,606 as described above. challenged with SP (10−8–10−6 M) for 24h, This 24-h stimulation time was chosen to ensure maximal cytokine release, as observed previously[12]. In some experiments, cells were pretreated with CP96,345 (1μM) or L703,606 (1μM) for 15 min before SP stimulation[13].The medium from Kupffer cells culture was collected and centrifuged at 1000 g for 5 min, and supernatant was kept -70℃ until assayed. IL-6 and TNF-α levels in the supernatant were determined with mouse-specific enzyme-linked immunosorbent assay (ELISA) kits. All samples, including standard and control solution, were assayed in duplicate. The measurements were performed according to the manufacturer's instructions. No cross-reactivity was observed with any other known cytokine. Results are expressed in picograms per milliliter. Statistical analysis All data were presented as means ± SEM. Differences among groups were assessed by using unpaired Student t-test and one-way analysis of variance. P-values of less than 0.05 were considered to be statistically significant. Calculations were performed with the SPSS, Version 11.0, statistical software package. Results. Detection of sp and liver function As shown in Figure 1, the levels of sp were significantly increased in the model group when compared with the control group (Figure 1A, 387.23 ± 29.36 vs 138.52 ±13.23pg/mL, P<0.05). The levels of serum ALT and AST were significantly increased in the model group when compared with the control group (782.37 ±21.51/1004.25 ± 18.24 U/L vs 42.05 ± 8.31/51.12 ± 9.16 U/L, respectively; P < 0.05). Compared with the model group, the levels of serum ALT and AST significantly decreased in the L-703,606 treated group (Figure 1B, 402.22 ±16.42/581.45 ± 17.51 U/L vs 782.37 ±21.51/1004.25 ± 18.24 U/L, respectively;P < 0.05). Figure 1. The levels of sp were significantly increased in groups 2 in comparison with groups 1(Figure 1A,), The ALT and AST levels were significantly increased in groups 2 in comparison with groups 1 and 3. Moreover, the ALT and AST levels were significantly decreased in group 3 compared to group 2 (Figure 1B).Results shown are the mean values ± SEM (n=12 mice per group), *P < 0.05 vs group 1, **P < 0.05 vs group 2. Histopathological changes Hepatic immune injury in mice was successfully established in this study. Histological observing showed that there are edema, hepatocellular apoptosis and granulocyte infiltration in livers of model group. Compared with control group(Figure 2A),model group mice liver tissue showed significant cytoplasmic vacuolization, sinusoidal congestion, extensive hepatic cellular necrosis and massive cellular infiltration (Figure 2B). However, the parenchymal appearance was near normal in L-703,606 group. Mild cellular infiltration, few necrosis as well as comparatively preserved lobular architecture were seen in the liver treated with L-703,606 (Figure 2C). Neurokinin-1 receptor antagonist L-703,606 can significantly alleviate concanavalin A-induced liver injury,It demonstrated that intervention of SP and its receptor had certain therapeutic effect on liver inflammation. Figure 2. Histopathological changes. There were no pathological changes in liver tissue of the group 1(Figure2A)(HE,×200). Extensive hepatic cellular necrosis and massive cellular infiltration were seen(Figure2B) (HE,×200). Decreased hepatic necrosis was seen compared to the Con A model group(Figure2C)(HE,×200). Mouse kupffer cells express NK1R and SP increases NK1RmRNA expression in KC in vitro NK1R in kupffer cells of mice were analyzed based on the immunohistochemical localization by fluorescein. Immunoreactivity was concentrated on the cell cytoplasm (Figure 3A), this has some difference with other studies.NK1R mRNA levels were upregulated in KC incubated with SP for 24 h. With the use of real-time PCR, the results further showed that SP increased NK1R mRNA expression 2.5 fold (Figure3B). NK1R blockade abolished the effect of SP on NK1R mRNA expression and significantly reduced NK1R mRNA expression compared with the control culture medium. Figure 3. (A) Nuclei were counterstained with DAPI (blue) and cells imaged using fluorescence microscopy. Immunoreactivity was concentrated on the cell cytoplasm (green).Merged fluorescent images are shown in the last one. Data are representative of three independent experiments. Data of negative controls are not shown.(B) Effects of SP and L-703,606 on neurokinin-1 receptor (NK-1R) mRNA expression in KC in vitro. The group data of NK1R mRNA are shown, Values are means ± SE; *P < 0.01 compared with CM. **P < 0.01 compared with SP. SP induces IL-6 and TNF-α production in a dose-dependent manner. To investigate the effect of different doses of SP on IL-6 and TNF-α synthesis in KC, KC were challenged at 37℃ with different concentrations of SP, ranging from 0.01 to 1uM(10−8-10−6M). IL-6/TNF-α levels were ranging from 75.11±7.13/36.21±6.42pg/ml respectively to 138.09±9.27/69.23±7.31pg/ml respectively. However the response reached a plateau when dose higher than 10−6 M, IL-6/TNF-α levels were 142.12± 11.09/72.15 ± 8.56 pg/ml respectively. A dose of 1μM (10−6M) SP was used to carry out subsequent experiments. Figure 4. SP induces IL-6 and TNF-α production in a dose-dependent manner in mouse KC. Results shown are means ± SE. SP enhances IL-6 and TNF-αsecreted and NK1R Receptor Antagonist Inhibits SP-induced IL-6 and TNF-αRelease from Kc in vitro To determine whether SP-induced IL-6 and TNF-αrelease is mediated through the NK-1 receptor, we preincubated kc with the NK-1 receptor antagonist L-703,606 (10μM) for 15 min and then during stimulation with the SP (1μM).As shown in Fig.5,The cytokine levels (IL-6/TNF-α) in supernatant from the release of cultured Kupffer cells revealed a substantial increase in the sp pretreated group as compared with the control culture medium group(142.12 ± 11.09/72.15 ± 8.56 pg/ml vs 71.13 ± 9.36/31.02 ± 7.26 pg/ml, respectively; P< 0.05). The IL-6/TNF-α levels of supernatant in L-703,606 pretreated group were significantly lower when compared with the sp pretreated group (68.16 ± 9.51/28.38 ± 5.04 pg/ml vs 142.12 ± 11.09/72.15 ± 8.56 pg/ml,respectively;P < 0.05).In other words, SP-induced IL-6 and TNF-α secretion from KC was abolished by pretreatment with L-703,606. Figure 5.Effects of SP and L703,606 on protein levels of interleukin (IL)-6 (A)and tumor necrosis factor (TNF)-α(B)secreted from KC in vitro. Values are means ± SE; *P < 0.05 compared with CM. **P < 0.05 compared with SP. Discussion. Concanavalin A (ConA)-induced hepatitis has long been used as an experimental model of immune-mediated liver disease[14],It is characterized by massive hepatocellular degeneration and lymphoid infiltration of the liver[15], In our study we established hepatic immune injury in mice successfully (Figure2B). Studies have demonstrated a pronounced influence by the autonomic nervous system on immune-mediated experimental hepatitis in the mouse[16].As is well-known, SP stimulates proinflammatory cytokine release ,SP has been shown to affect cytokine production/release from various immune cells, For example, SP can modulate the release of IL-1, IL-6, and TNF-α from human blood monocytes[11],In monocyte/macrophages, SP also stimulates the release of both arachidonic acid metabolites and proinflammatory cytokines, induces the respiratory burst and acts as a potent chemoattractant[17]. Pro-inflammatory signaling mediated by substance P acts through NK-1 receptors[18],In particular, the neurokinin-1 receptor (NK-1R) has been detected on lymphocytes, granulocytes, macrophages, and dendritic cells and probably also mast cells, and nonreceptor-mediated effects of SP and other tachykinins may also take place in various immunocytes[6,19,20]. Previous studies indicate that NK1 receptors are upregulated at inflamed sites in many tissues, including joints and intestine[21,22,23]. Release of the neuropeptide substance P and activation of its selective receptor, the NK1R, are associated with inflammation and pain such as acute pancreatitis[24]and endotoxemia[25]. It is also reported that SP and selective NK1 agonists induce superoxide anion production, tumour-necrosis factor (TNF)-α release in human monocytes and alveolar macrophages[26]. So because neurogenic inflammation can result from the action of substance P on NK1 receptors, it is of interest to study the effect of blockage of selective NK1 receptor using specific NK-1 antagonist would decrease the injury. L703,606 is selective NK-1R antagonists, Importantly, studies have verified that the effects of L703,606 are attributable to pharmacological blockade of endogenous SP/NK-1R interactions in parallel studies using NK-1R−/−animals.In this study, we observed that pretreatment with Neurokinin-1 receptor antagonist L-703,606 significantly improved liver function and ameliorated hepatic injury. So it was concluded that the therapeutic potential of SP receptor (neurokinin-1 receptor) antagonists should not be ignored. We speculate that this protective effect may act in part via the reduction in cytokine levels. Cytokines serve as central regulators controlling genes, responsible for either inducing apoptosis or protective action on cell by stimulating proliferation of hepatocyte. So we tested whether SP+LPS evoked kupffer cells secretion of IL-6 and TNF-α in vitro. Kupffer cells (KCs) are non-parenchymal cells that account for approximately 15% of the total liver cell population and constitute 80%–90% of the tissue-resident macrophages in the whole body[27,28]. Kupffer cells represent the major source of inflammatory cytokine production and thus systemic release of proinflammatory mediators[29].Multiple lines of evidence have suggested that they are critical to the onset of liver injury and following secretion, cytokines aggravate hepatocyte damage. The use of in vitro and animal models has shown that inactivation of Kupffer cells prevents liver injury[30].Actived Kupffer cells results in the release of an array of inflammatory mediators, growth factors, and reactive oxygen species such as TNF-α and IL-6 which contribute to hepatocellular damage[31]. SP has been detected within liver and receptors for SP have also been detected on Kupffer cell and hepatocytes. However, there is limited information on the relations between sp and cytokines in Kupffer cell. Our studies have a conclusion that mouse kupffer cells expressed NK1R and SP increased NK-1RmRNA expression in KC in vitro. We report in this work for the first time that Sp and LPS induces IL-6 and TNF-α production from kupffer cells in a dose-dependent manner. a dose >10−6 M the response reached a plateau. This dose was found to effectively activate the cells and induce a rise in chemokine release, whereas lower doses were considerably less effective. At the same concentration, SP is reported to stimulate significant chemokine production by pancreatic acinar cells and prime neutrophils triggered by different stimuli to evoke various cellular responses including intracellular calcium changes, oxidative responses, and the formation of hydrogen peroxide and nitric oxide[32,33]. In summary, the present and our previous studies indicate that inflammatory cytokine-mediated apoptotic liver injury is affected by neuropeptides. NK-1R agonists such as SP seem to be major players in this scenario by up-regulating the proinflammatory cytokine response. If it turns out that data from our mouse experiments on the hepatoprotective potential of NK-1R antagonists can be transferred to clinical practice, this may represent a major advance in the treatment of hepatitis. References 1. Richardson JD, Vasko MR. Cellular mechanisms of neurogenic inflammation. J pharmacol Exp Ther 2002;302:839–845. 2. Sternberg EM. Neural regulation of innate immunity: a coordinated nonspecific host response to pathogens. Nat Rev Immunol 2006;6:318–328. 3. Satake H, Kawada T. Overview of the primary structure, tissue-distribution, and functions of tachykinins and their receptors. Curr Drug Targets 2006;7:963–974. 4. Greco SJ, Corcoran KE, Cho KJ, Rameshwar P. 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