Download In our study we established hepatic immune injury in mice successfully

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.
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