The Journal of Immunology
The Role of Neutral Endopeptidase in Caerulein-Induced
Yung-Hua Koh,* Shabbir Moochhala,*,†and Madhav Bhatia‡
Substance P (SP) is well known to promote inflammation in acute pancreatitis (AP) by interacting with neurokinin-1 receptor.
However, mechanisms that terminate SP-mediated responses are unclear. Neutral endopeptidase (NEP) is a cell-surface enzyme
that degrades SP in the extracellular fluid. In this study, we examined the expression and the role of NEP in caerulein-induced AP.
Male BALB/c mice (20–25 g) subjected to 3–10 hourly injections of caerulein (50 mg/kg) exhibited reduced NEP activity and
protein expression in the pancreas and lungs. Additionally, caerulein (1027M) also downregulated NEP activity and mRNA
expression in isolated pancreatic acinar cells. The role of NEP in AP was examined in two opposite ways: inhibition of NEP
(phosphoramidon [5 mg/kg] or thiorphan [10 mg/kg]) followed by 6 hourly caerulein injections) or supplementation with exog-
enous NEP (10 hourly caerulein injections, treatment of recombinant mouse NEP [1 mg/kg] during second caerulein injection).
Inhibition of NEP raised SP levels and exacerbated inflammatory conditions in mice. Meanwhile, the severity of AP, determined
by histological examination, tissue water content, myeloperoxidase activity, and plasma amylase activity, was markedly better in
mice that received exogenous NEP treatment. Our results suggest that NEP is anti-inflammatory in caerulein-induced AP. Acute
inhibition of NEP contributes to increased SP levels in caerulein-induced AP, which leads to augmented inflammatory responses in
the pancreas and associated lung injury. The Journal of Immunology, 2011, 187: 5429–5439.
billion dollars in hospitalization costs (1, 2). In ∼80% of cases,
patients suffer mild symptoms and usually recover in a few days,
whereas others experience a severe attack with a high mortality
rate (2). The primary cause of mortality in severe AP is necrosis
of the pancreas, often followed by systemic inflammatory re-
sponse syndrome, which causes injuries to distant organs such as
lungs and kidneys (3–5). Heavy alcohol consumption and gall-
stones are two main etiologies of severe AP, but up to 20% remain
idiopathic (2). It is generally accepted that premature activation of
zymogens in pancreatic acinar cells leads to autodigestion of the
organ, causing subsequent liberation of proinflammatory media-
tors that intensify the inflammatory responses (6). Despite recent
advances in understanding the pathogenesis of AP, cellular medi-
ators that determine the severity of AP are complex and incom-
Substance P (SP) is widely held to exert its effects on target
cells expressing neurokinin-1 receptor (NK1R). SP–NK1R inter-
cute pancreatitis (AP) is the sudden inflammation of the
pancreas. In the United States, ∼210,000 patients seek
treatment for AP annually, placing a huge burden of .2
action plays an early and important role in the inflammatory
cascade and promotes excessive activation of inflammatory cells.
Proinflammatory mechanisms of SP are thought to be its effects on
plasma extravasation, neutrophil recruitment, and inflammatory
mediator synthesis upon exogenous stimuli (7, 8). SP is encoded
by the preprotachykinin-A (PPTA) gene, produced primarily by
sensory nociceptive neurons, but it can also be produced by in-
flammatory cells and pancreatic acinar cells (9–11). SP–NK1R
interaction was shown to be a key mediator in the pathogenesis of
experimental AP. Pharmacological antagonism of NK1R, knock-
out of PPTA gene, or disruption of SP release from the nerve
endings protected mice against AP and associated lung injury (12–
14). A rapid increase in SP levels and gene expression was ob-
served in caerulein-treated mice or caerulein-treated murine pan-
creatic acinar cells (13, 15). Early upregulation of SP expression
positions it to influence many of the early inflammatory responses.
Increased expression of SP in SP-expressing cells and increased
release of SP from nerve endings may contribute to elevated SP
levels in experimental AP. However, inhibitory mechanisms that
terminate the effect of SP were not clearly understood. The cell-
surface enzyme neutral endopeptidase (NEP; also called enkepha-
linase, neprilysin, common acute lymphoblastic leukemia Ag, or
CD10) is known to degrade a variety of short peptides in the ex-
tracellular fluid (16, 17). NEP is capable of modulating inflam-
matory responsesbydegradation of SP (18,19). Thisissupported
by studies showing that NEP knockout or inhibition potentiates
inflammation, but was prevented by cotreatment with NK1R
antagonists (18). Additionally, administration of exogenous recom-
such as intestinal inflammation and burns (18, 20, 21). Current ev-
idence supports that NEP plays an anti-inflammatory role.
To date, the role of NEP in the pathogenesis of AP is not
thoroughly understood. Genetic deletion of NEP exacerbated pan-
creatitis-associated lung injury and greatly increased mortality rate
in choline-deficient and ethionine-supplemented diet-induced AP
(22). However, physiological changes of NEP expression and
*Department of Pharmacology, National University of Singapore, Singapore 117597;
†Defence Medical and Environmental Research Institute, DSO National Laboratories,
Singapore 117510; and‡Department of Pathology, University of Otago, Christchurch
8140, New Zealand
Received for publication July 12, 2011. Accepted for publication September 13,
This work was supported by National Medical Research Council Grant R-184-000-
Address correspondence and reprint requests to Prof. Madhav Bhatia, Department of
Pathology, University of Otago, 2 Riccarton Avenue, P.O. Box 4345, Christchurch
8140, New Zealand. E-mail address: firstname.lastname@example.org
Abbreviations used in this article: AP, acute pancreatitis; MPO, myeloperoxidase;
NEP, neutral endopeptidase; NK1, neurokinin-1; PPTA, preptotachykinin-A; SP, sub-
stance P; Suc-Ala-Ala-Phe-pNA, succinyl-Ala-Ala-Phe-p-nitroanilide.
activity in caerulein-induced AP are not known. Therefore, in the
present study, we investigated the potential regulatory role of
caerulein on NEP activity and expression. Subsequently, NEP in-
hibitors and exogenous NEP were used to examine the effects of
NEP on the outcome of AP. Although the primary focus of this
article is pancreatic injury, lung injury is often observed in severe
AP (13, 22). Hence, lungs have also been investigated for path-
Materials and Methods
Animals and chemicals
All experimental procedures were approved by the Animal Ethics Com-
with established International Guiding Principles for Animal Research.
BALB/c mice (male, 20–25 g) were acclimatized in a controlled envi-
ronment with an ambient temperature of 23˚C and a 12-h light/dark cycle.
Caerulein was purchased from Bachem (Torrance, CA). Glucose, HEPES,
and soybean trypsin inhibitor were obtained from Sigma-Aldrich (St.
Louis, MO). Type IV collagenase was purchased from Worthington Bio-
chemical (Freehold, NJ). Recombinant mouse NEP was purchased from
Sino Biological (Beijing, China). Phosphoramidon and thiorphan (NEP
inhibitors) were purchased from Sigma-Aldrich. All chemicals were pur-
chased with the highest purity available.
Preparation of pancreatic acini
Pancreatic acini were obtained from mouse pancreas by collagenase
treatment as described previously (11). Briefly, mice were euthanized by
a lethal dose of sodium pentobarbitone (150 mg/kg). Fresh pancreas was
infused with buffer A (140 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl2,
1 mM CaCl2, 10 mM glucose, 10 mM HEPES, and 0.5 mg/ml soybean
trypsin inhibitor [pH 7.3]) containing 200 IU/ml type IV collagenase. The
pancreas was then minced and digested with buffer A containing 200 IU/
ml type IV collagenase (10 min, 37˚C). To obtain dispersed acini, the
digested tissue was passed through small pipette tips. The viability of
pancreatic acinar cells was determined by trypan blue exclusion assay. Cell
preparations with at least 95% viability were used for further experiments.
Isolated pancreatic acinar cells were treated with caerulein (1027M) for
0–120 min for investigation of NEP activity and mRNA expression.
Induction of AP
BALB/c micewere randomly assigned to control or experimental groups by
using 10 animals for each group. Animals were given hourly i.p. injections
of saline containing caerulein (50 mg/kg) for 3, 6, or 10 h to induce mild,
moderate, or severe AP, respectively (in terms of relative severity of
caerulein-induced models). Control mice received hourly normal saline
injections. To investigate whether NEP inhibition exacerbates AP, mice
received a pretreatment of phosphoramidon (5 mg/kg, i.v.) or thiorphan
(10 mg/kg, i.v.) 1 h before six hourly caerulein injections. These concen-
trations were chosen in reference to previous studies (23, 24). Additionally,
to investigate the protective effects of NEP, mice received 10 hourly
caerulein injections and received posttreatment of recombinant mouse
NEP (1 mg/kg, i.v.) during the second injection of caerulein. One hour
after the last caerulein injection, animals were killed by a lethal dose of
sodium pentobarbitone (150 mg/kg, i.p.). Samples of pancreas, lung, and
blood were collected. Plasma was prepared from anticoagulated blood
samples by centrifugation (10,000 3 g, 5 min, 4˚C). Random cross-
sections of the head, body, and tail of the pancreas and samples of the right
lung were fixed in 10% neutral buffered formalin (Sigma-Aldrich). A small
section of pancreas and lung was weighed and then dried for 72 h at 55˚C
and reweighed to determine pancreatic water content. Remaining samples
were then stored at 280˚C for subsequent analysis.
Measurement of NEP activity
NEP enzymatic activity was determined spectrophotometrically from
extracted protein samples as described previously (25). Briefly, treated
pancreatic acinar cells, pancreas, and lung tissue were homogenized in T-
PER tissue protein extraction reagent (Pierce, Rockford, IL) and centri-
fuged (12,000 3 g, 15 min, 4˚C). Cell-free extracts (30 mg protein) were
incubated with 1 mM succinyl-Ala-Ala-Phe-p-nitroanilide (Suc-Ala-Ala-
Phe-pNA; Bachem) as a substrate in 0.1 M Tris-HCl (pH 7.6) in the
presence of 1 U porcine kidney aminopeptidase (AP-N; Sigma-Aldrich).
In this coupled activity assay, NEP cleaves Suc-Ala-Ala-Phe-pNA between
Ala and Phe, yielding Phe-pNA. AP-N subsequently cleaves Phe-pNA,
generating pNA as the final product. The kinetic change in absorbance
at 405 nm due to the accumulation of free pNA was determined each
minute at 37˚C for 60 min using a microplate reader. Substrate alone and
substrate with AP-N and Tris buffer blanks were run in parallel. The rate of
absorbance change were normalized with the control and expressed as fold
change to control. Protein concentrations were determined by a Bradford
protein assay (26).
SP extraction and detection
Pancreas and lung tissues were homogenized in 1 ml ice-cold SP assay
buffer (Bachem). The homogenates were centrifuged (13,000 3 g, 20 min,
4˚C) and the supernatants collected. They were adsorbed on C18cartridge
columns (Bachem) as previously described and eluted with 1.5 ml 75%
acetonitrile (27). Samples were freeze-dried overnight and reconstituted
with SP assay buffer. SP content was then determined with an ELISA kit
(Bachem) according to the manufacturer’s instructions. The results were
quantified by spectophotometry at 450 nm. The results were then nor-
malized with DNA content of the tissue samples fluorometrically by using
Hoechst dye 33256 and calf thymus DNA as a standard (28). SP expression
was corrected as nanograms per microgram of DNA.
Plasma amylase activity was measured by using a kinetic spectrophoto-
metric assay according to the manufacturer’s instructions (Thermo Fisher
Scientific, Singapore). Plasma samples were incubated with Infinity am-
ylase CNPG3liquid stable reagent (Thermo Fisher Scientific) for 1 min at
37˚C, and absorbance was measured every minute for the subsequent 5 min
at 405 nm. The change in absorbance was used to calculate the amylase
Measurement of myeloperoxidase activity
Neutrophil sequestration in the pancreas and lung was quantified by
measuring tissue myeloperoxidase (MPO) activity. Tissue samples were
thawed, homogenized in 20 mM phosphate buffer (pH 7.4), centrifuged
(13,000 3 g, 10 min, 4˚C), and the resulting pellet was resuspended in
50 mM phosphate buffer (pH 6.0) containing 0.5% (w/v) hexadecyl-
trimethylammonium bromide (Sigma-Aldrich). The suspension was sub-
jected to four cycles of freezing and thawing and was further disrupted by
sonication (40 s). The sample was then centrifuged (13,000 3 g, 5 min,
4˚C) and the supernatant used for MPO assay. The reaction mixture con-
sisted of the supernatant (50 ml), 1.6 mM tetramethylbenzidine (Kirke-
gaard & Perry Laboratories, Gaithersburg, MD), 80 mM sodium phosphate
buffer (pH 5.4), and 0.3 mM hydrogen peroxide (reagent volume, 50 ml).
This mixture was incubated at 37˚C and terminated with 50 ml 0.18 M
H2SO4. The absorbance was measured at 450 nm. This absorbance was
then corrected for the DNA content of the tissue sample and results were
expressed as fold change over control (28).
A small portion of pancreas was excised and fixed with 10% neutral
buffered formalin (Sigma-Aldrich), dehydrated through a graded ethanol
thickness were stained with H&E and examined by light microscopy using
a Nikon Eclipse 80i microscope (objective lens magnification 320; eye-
piece magnification 310).
ELISA assays were performed for the measurement of cytokines (IL-1,
IL-6, TNF-a), chemokines (MIP-1a, MIP-2), and adhesion molecules
(ICAM-1,VCAM-1, P-selectin, E-selectin) in pancreas and lung tissue
homogenates, according to the manufacturer’s instructions (R&D Systems,
Minneapolis, MN). Results were then corrected for the DNA content of
the tissue homogenates and were expressed as picograms per microgram
of DNA (28). In this study, tissue content of DNA was chosen over
tissue weight or tissue protein content as a normalization factor in ELISA
analysis. In the pancreas, it has been shown that total DNA content (mil-
ligrams DNA per pancreas) remained relatively stable, but total protein
content (milligrams protein per pancreas) was decreased in caerulein-
induced pancreatitis models (29). Pancreatic edema in AP also alters the
proportion of water in the tissue, which might cause bias when total
pancreatic weight was used as a normalization factor.
Whole cell lysate preparation and Western blot analysis
Pancreatic tissue was homogenized in ice-cold radioimmunoprecipitation
assay lysis buffer supplemented with protease inhibitor mixture (Sigma-
5430PROTECTIVE ROLE OF NEP IN CAERULEIN-INDUCED AP
Aldrich) and phosphatase inhibitor mixture (Sigma-Aldrich). Protein con-
centrations were determined by Bradford protein assay. Western blotting
was performedusing rabbit monoclonalanti-NEP (1:2500 dilution,Abcam)
or hypoxanthine-guaninephosphoribosyltransferase (1:1000dilution;Santa
Cruz Biotechnology) in a buffer containing 2.5% nonfat dry milk in 0.05%
Tween 20 in PBS. Afterwards, the membranes were incubated with goat
anti-rabbit HRP-conjugated secondary Ab (1:2000 dilution; Santa Cruz
Biotechnology). Membranes were washed and then incubated in Super-
Signal West Pico chemiluminescent substrate (Pierce) before exposure
to x-ray films (CL-Xposure; Pierce). Hypoxanthine-guanine phosphor-
ibosyltransferase was applied as an internal control to normalize protein
loading. The intensity of bands was quantified using LabWorks image
analysis software (UVP).
Quantitative real-time PCR analysis
Total RNA from snap-frozen pancreatic tissue was extracted with TRIzol
reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s pro-
tocol. All the steps were done in ice-cold conditions. The integrity of RNA
was verified by ethidium bromide staining for the presence of distinct 28S
and 18S bands on a 1.2% agarose gel. One microgram total RNA was
reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad, Her-
cules, CA) in a total volume of 20 ml, according to the manufacturer’s
instructions. cDNA (2 ml) was used as a template for PCR amplification by
using a SYBR Green PCR Master Mix from Roche Diagnostics (Singa-
pore). No template controls and intron spanning primers were used. All
reactions were done in duplicates. PCR reaction mix was first subjected to
95˚C for 5 min, followed by 45 cycles of amplification. Each cycle con-
sisted of 95˚C for 30 s, annealing temperature of 55˚C for 15 s, and
elongation temperature of 72˚C for 15 s. b-actin was used as a house-
keeping gene to normalize the mRNA expression. Expression of NEP,
PPTA, NK1R, and b-actin was determined using the “crossing point” of
the sample, which is the point (cycle number) at which the fluorescence of
a sample rises above the background fluorescence.
The data are expressed as means 6 SEM, and the absence of such bars
indicates that the SE is too small to illustrate. The significance of differ-
ence among groups was evaluated by ANOVA with Tukey’s post hoc test
for multiple comparisons when comparing three or more groups. A p value
, 0.05 was regarded as statistically significant.
Caerulein suppress NEP activity and mRNA expression in
isolated pancreatic acinar cells
Pancreatic acinar cell NEP activity was significantly decreased
as early as 30 min after treatment with caerulein (1027M), when
compared with the control. A time-dependent decrease of NEP
enzymatic activity was revealed, reaching a minimum after 2 h
caerulein stimulation (Fig. 1A). Moreover, NEP mRNA expression
was significantly downregulated in pancreatic acinar cells that
were stimulated with caerulein for .60 min (Fig. 1B, Table I).
Cells that were incubated in control buffer did not show a signif-
icant change in both NEP activity and mRNA expression (Fig. 1A,
Caerulein-induced AP suppress endogenous NEP activity
The suppression of NEP activity in caerulein-treated pancreatic
could be reproduced in animal models of AP. BALB/c mice were
given hourly i.p. injections of caerulein for 3, 6, or 10 h to induce
mild, moderate, or severe AP, respectively, in terms of relative
severity. Caerulein-treated mice showed a rapid decrease of
(1027M) for 0–120 min. A, Time-dependent decrease of NEP activity in pancreatic acinar cells. B, NEP mRNA expression in pancreatic acinar cells.
Values are means 6 SEM; n = 4–6 mice/time point. Mice received hourly caerulein injections (50 mg/kg). C, Pancreas NEP activity. D, Pancreas NEP
protein expression. E, Lung NEP activity. F, Lung NEP protein expression. Values are means 6 SEM; n = 10 mice/time point. *p , 0.05 versus control.
Administration of caerulein decreased NEP activity and expression. Freshly prepared pancreatic acinar cells were treated with caerulein
The Journal of Immunology 5431
pancreas NEP activity, with maximal suppression observed at
severe AP conditions (Fig. 1C). Consistent with NEP activity
results, Western blot analysis confirmed early and sustained de-
crease of NEP protein expression in the pancreas (Fig. 1D). In the
lungs, similar decreases of NEP activity and protein expression
were observed after induction of AP (Fig. 1E, 1F). Notably,
protein expression of NEP in the lung showed a large magnitude
of decrease when compared with the control (Fig. 1F).
Phosphoramidon and thiorphan increase SP levels in the
pancreas, lung, and plasma
As NEP was previously shown to be protective against excessive
inflammation, we anticipated that further suppression of NEP ac-
tivity should exaggerate inflammatory conditions in AP (18, 19).
Therefore, we preinhibited NEP before induction of moderate AP
to investigate whether it would degenerate into a more severe form
of AP. To do this, mice received a single dose of phosphoramidon
(5 mg/kg, i.v.) or thiorphan (10 mg/kg, i.v.), followed by six hourly
caerulein or saline injections. Both NEP inhibitors decreased NEP
activity in the pancreas by ∼70% 1 h posttreatment (data not
shown). Control mice challenged with a single dose of phos-
phoramidon or thiorphan still showed a 20–30% reduction of NEP
activity in the pancreas and lungs after six hourly saline injections,
suggesting metabolism and clearance of the drug over time (Fig.
2A, 2C). In contrast, SP levels rose significantly (Fig. 2B, 2D).
Induction of moderate AP suppressed NEP activity in both pan-
creas and lungs, coupled with a strong increase of SP in the
pancreas and a modest but insignificant increase in the lungs
phoramidon (5 mg/kg, i.v.) or thiorphan (10 mg/kg, i.v.) before six hourly injections of caerulein (50 mg/kg, i.p.) or normal saline. One hour after the last
caerulein injection, mice were sacrificed and NEP activity and SP ELISA assays were performed as described in Materials and Methods. A, Pancreas NEP
activity. B, Pancreas SP levels. C, Lung NEP activity. D, Lung SP levels. E, Plasma NEP activity. F, Plasma SP levels. Values are means 6 SEM; n = 10
mice/time point. *p , 0.05 versus control,#p , 0.05 versus caerulein group.
Inhibition of NEP by phosphoramidon and thiorphan decreased NEP activity and increased SP levels. Mice were randomly given phos-
Table I.PCR primer sequences and product sizes
Gene Primer SequenceSize (bp) Nucleotide Accession No.
Nucleotides are available at: http://www.ncbi.nlm.nih.gov/nuccore/.
5432 PROTECTIVE ROLE OF NEP IN CAERULEIN-INDUCED AP
(0.10 . p . 0.05). Interestingly, pretreatment of NEP inhibitors
followed by induction of AP provoked a further increase in SP
levels (Fig. 2B, 2D) but did not further reduce NEP activity (Fig.
2A, 2C) in pancreas and lungs. Phosphoramidon or thiorphan
administration did not alter background NEP activity in plasma,
as NEP is a membrane-bound enzyme and not normally found in
cell-free plasma (Fig. 2E). Despite this, plasma SP concentrations
were significantly elevated in all mice that received NEP inhibitor
treatments when compared with their respective untreated controls
Effect of NEP inhibition on plasma amylase activity, MPO
activity, tissue water content, and pancreatic histology
After confirming that phosphoramidon and thiorphan elevated SP
levels in mice, we assessed how NEP inhibition influences the
outcome of AP. Amylase is produced in the pancreatic acinar cells
and released into the bloodstream when there is a pancreatic injury,
and therefore it is often used clinically as a marker for the diagnosis
of AP. Another important feature of AP is an elevated MPO activity
in the pancreas and lungs, indicating infiltration of neutrophils
into these tissues (27). Inhibition of NEP without induction of AP
did not alter basal plasma amylase activity, tissue water content,
or tissue MPO activity, despite elevated SP levels (Fig. 3). Ad-
ditionally, NEP inhibitors further elevated plasma amylase activ-
ity, pancreas water content, and pancreas MPO activity in AP-
induced mice (Fig. 3A–C). The lungs were less affected, as NEP
inhibitors did not alter AP-induced increases in lung MPO activity
and water content (Fig. 3D, 3E). Histological examination of the
pancreas confirmed that inhibition of NEP worsened damage in
AP-induced mice, characterized by increased pancreatic edema,
neutrophil infiltration, and pancreatic necrosis (Fig. 4B, 4D, 4F).
Normal pancreas architecture was observed in all control mice
(Fig. 4A, 4C, 4E).
Effect of NEP inhibition on proinflammatory cytokine,
chemokine, and adhesion molecule expression
Mice induced with moderate AP exhibited a significant elevation
in pancreatic proinflammatory mediators, which include cytokines
(IL-1, IL-6, TNF-a) and chemokines (MIP-1a, MIP-2) (Table II),
compared with control mice, which received saline injections.
Adhesion molecule (ICAM-1, VCAM-1, P-selectin, E-selectin)
expression also showed modest increases, but they were not sig-
nificantly different from the control. In the lungs, a milder up-
regulation of proinflammatory mediators was observed, with only
IL-1 and ICAM-1 significantly increased from control mice (Table
II). Inhibition of NEP in AP-induced mice generally showed a
further increase of proinflammatory mediator expression in both
pancreas and the lungs when compared with the control groups
(Table II). NEP inhibitors alone did not appear to raise tissue
expression of any of the investigated molecules.
Mouse recombinant NEP decreases SP levels in the pancreas,
lung, and plasma
We have observed suppression of NEP activity in caerulein-
induced AP. Therefore, we investigated whether mice would be
protected against severe AP by treatment of exogenous NEP. Mice
that received 10 hourly caerulein injections developed severe AP
and exhibited significantly suppressed NEP activity in the pan-
creas and lungs (Fig. 5A, 5C). Administration of exogenous NEP
markedly increased NEP activity as well as decreased SP levels in
the lung and pancreas (Fig. 5B, 5D). Induction of AP did not affect
the background plasma NEP activity, but a 10-fold increase was
observed after mice were treated with exogenous NEP (Fig. 5E).
Treatment of mice with exogenous NEP did not completely
abolish pathological SP levels in the pancreas and plasma. SP
levels were still significantly higher in NEP-treated AP mice when
compared with the control (Fig. 5B, 5F).
ramidon (5 mg/kg, i.v.) or thiorphan (10 mg/kg, i.v.) before six hourly injections of caerulein (50 mg/kg, i.p.) or normal saline. One hour after the last
caerulein injection, mice were sacrificed and amylase activity, MPO activity, and water content measurement were performed as described in Materials and
Methods. A, Plasma amylase activity. B, Pancreas MPO activity. C, Pancreas water content. D, Lung MPO activity. E, Lung water content. Values are means 6
SEM; n = 10 mice/time point. *p , 0.05 versus control,#p , 0.05 versus caerulein group.
Effect of NEP inhibition on plasma amylase activity, tissue MPO activity, and tissue water content. Mice were randomly given phospho-
The Journal of Immunology 5433
Exogenous NEP protects mice against caerulein-induced
Plasma amylase, as well as MPO activity and water content in
the pancreas and lungs, was strongly augmented after induction of
severe AP (Fig. 6). Mouse recombinant NEP (1 mg/kg, i.v.), given
during the second caerulein injection, significantly attenuated AP-
induced MPO activity and water content (Fig. 6B–E). Exogenous
NEP also protected mice against pancreatic injury, as shown by
a decrease in plasma amylase activity (Fig. 6A). Histological ex-
amination of the pancreas confirmed the protective effects, as
demonstrated by reduced neutrophil infiltration, pancreatic edema,
and pancreatic necrosis (Fig. 4G, 4H). Notably, the therapeutic
effect of exogenous NEP correlated with a decrease of SP levels.
However, a single dose of exogenous NEP treatment did not
completely protect mice from severe AP, as pancreatic morphol-
ogy, plasma amylase activity, and tissue MPO activity were still
significantly increased when compared with healthy control mice.
Effect of exogenous NEP treatment on proinflammatory
cytokine, chemokine, and adhesion molecule expression
Mice that received 10 hourly caerulein injections (severe AP)
showed a further increase of proinflammatory mediator levels in
pancreas and lung tissue when compared with mice that received 6
hourly caerulein injections (moderate AP) (Tables II, III). Notably,
NEP treatment decreased expression of proinflammatory cyto-
kines (IL-1, IL-6, TNF-a), chemokines (MIP-1a, MIP-2), and
adhesion molecules (ICAM-1, VCAM-1, E-selectin, P-selectin) in
mice induced with severe AP (Table III). Consistent with MPO
and histology results, exogenous NEP treatment did not com-
pletely protect mice from AP-induced upregulation of cytokines,
chemokines, and adhesion molecules (Table III).
Exogenous NEP attenuates caerulein-induced NK1R mRNA
upregulation in the pancreas
It was previously reported that disruption of SP–NK1R interaction
by CP96,345, a selective NK1R antagonist, decreased PPTA and
NK1R mRNA expression in AP (30). Therefore, in this study, we
investigated the effect of NEP on mRNA expression of PPTA,
NK1R, and NEP in the pancreas. Expectedly, we observed up-
regulation of PPTA and NK1R mRNA expression after induction
of AP (Fig. 7C–F, Table I). However, we found that only NK1R
expression was downregulated when mice with severe AP re-
ceived exogenous NEP treatment (Fig. 7F). Inhibition of NEP by
phosphoramidon or thiorphan did not affect caerulein-induced
PPTA/NK1R upregulation (Fig. 7C, 7E). Interestingly, NEP
mRNA expression was significantly decreased in moderate AP, but
recovered to normal levels in severe AP (Fig. 7A, 7B).
A great deal of preclinical data and some early clinical studies
highlighted the importance of tachykinin interaction with their
receptors for promoting inflammatory disorders (18, 19, 25, 31,
32). Thus, identifying the molecular mechanisms that modulate
their expression is of crucial importance. Our earlier work has
identified the mechanisms that lead to upregulation of SP in the
pancreatic acinar cells following caerulein stimulation (11, 15). In
the present study, we report that caerulein-induced AP suppressed
NEP activity in the pancreas and lungs, which could contribute to
elevated SP levels in the system and promote subsequent inflam-
matory responses. We also report that inhibition of NEP with
phosphoramidon or thiorphan exacerbated inflammation and, for
the first time to our knowledge, that treatment of AP-induced mice
with exogenous NEP protected mice against severe AP.
Our findings identified that NEP was downregulated by caer-
ulein in mouse and isolated mouse pancreatic acinar cells. Time
course studies revealed an early and rapid decrease of NEPactivity
after induction of AP, suggesting that NEP might be involved in
early inflammatory responses. Besides, sustained downregulation
of NEP activity throughout the course of AP could contribute to
uncontrolled inflammation. Interestingly, whereas pancreatic NEP
mRNA expression remained downregulated during moderate AP,
its expression during severe AP recovered to a level comparable
to normal, healthy mice. The effects of longer exposure to
caerulein on pancreatic NEP expression might suggest activation
of recovery mechanisms that help to reverse the ongoing inflam-
Although we showed a downregulation of NEP activity in
caerulein-induced AP, the mechanisms that regulate its expression
polymorphonuclear leukocyte infiltration and injury. Formalin-fixed tissue
sections were embedded in wax and cut into 5-mm sections. A, Control. B,
Mice received six hourly caerulein (50 mg/kg) injections. C, Mice received
a single dose of phosphoramidon (5 mg/kg) followed by saline injections.
D, Mice received a single dose of phosphoramidon (5 mg/kg) followed by
six hourly caerulein injections. E, Mice received a single dose of thiorphan
(10 mg/kg) followed by saline injections. F, Mice received a single dose of
thiorphan (10 mg/kg) followed by six hourly caerulein injections. G, Mice
received 10 hourly caerulein injections. H, Mice received a single dose of
exogenous NEP (1 mg/kg) and 10 hourly caerulein injections. Scale bars,
Histopathological evaluation (H&E staining) of pancreas
5434 PROTECTIVE ROLE OF NEP IN CAERULEIN-INDUCED AP
remains unclear. Caerulein might directly inactivate transcription
factors that regulate NEP mRNA expression. However, this did not
adequately address why downregulation of NEP activity occurred
before downregulation of NEP mRNA in the pancreatic acinar
cells. Activation of proteases, necrosis of pancreatic tissue, or gen-
eration of reactive oxygen species might also indirectly reduce
NEP activity in caerulein-induced models (33). Oxidative stress
was shown to inactivate NEP in the lungs (34, 35). Downregula-
tion of NEP was also observed in inflammatory diseases inde-
pendent of caerulein stimulation, such as inflamed rat intestine and
Table II. Effect of NEP inhibition on expression of cytokines, chemokines, and adhesion molecules
ControlCae6h Phos + Cae6h Thior + Cae6h PhosThior
Expression in pancreas
Expression in lung
3.15 6 0.22
4.08 6 0.27
6.62 6 0.40
2.21 6 0.29
4.11 6 0.24
8.10 6 0.57
26.36 6 1.99
33.96 6 3.11
35.63 6 2.19
5.79 6 0.4*
5.98 6 0.61*
10.54 6 0.68*
3.67 6 0.16*
6.36 6 0.58*
9.33 6 0.81
31.15 6 3.52
41.42 6 3.05
38.32 6 3.16
6.41 6 0.37*
7.98 6 0.50*,#
12.1 6 0.66*,#
6.00 6 0.39*,#
8.77 6 0.52*,#
12.25 6 1.20*
43.55 6 4.63*
48.39 6 5.69
59.30 6 7.26*
7.38 6 0.41*,#
7.74 6 0.63*
11.63 6 0.49*,#
5.46 6 0.53*,#
6.68 6 0.50*
11.12 6 1.08
41.42 6 4.03*
52.51 6 5.28*
48.49 6 5.49
4.35 6 0.29
4.1 6 0.43
7.38 6 0.45
2.85 6 0.27
5.18 6 0.48
10.19 6 0.94
33.42 6 2.28
42.51 6 4.98
35.49 6 2.46
3.93 6 0.36
4.42 6 0.31
7.70 6 0.69
2.47 6 0.27
5.22 6 0.54
7.87 6 0.97
26.60 6 3.19
40.31 6 4.71
38.07 6 4.48
13.52 6 1.12
11.91 6 0.53
10.00 6 0.94
6.87 6 0.44
11.16 6 0.42
21.92 6 1.35
140.4 6 13.9
218.3 6 12.5
199.0 6 16.3
21.54 6 2.25*
13.57 6 2.05
16.34 6 2.49
9.76 6 0.76
14.35 6 1.36
26.39 6 2.88
168.5 6 15.7
366.0 6 21.6*
220.0 6 9.7
20.04 6 2.52
14.07 6 1.40
20.94 6 1.84*
12.56 6 1.75*
17.22 6 2.11*
29.60 6 4.63
193.9 6 22.7
416.3 6 55.9*
249.0 6 32.6
18.21 6 1.52
12.34 6 1.36
18.69 6 2.28*
10.57 6 1.44*
15.77 6 1.45*
25.02 6 2.94
154.0 6 18.8
369.7 6 44.5*
184.2 6 14.3
12.81 6 1.65
9.78 6 1.44
13.17 6 2.28
6.85 6 0.39
11.69 6 1.30
18.43 6 1.93
142.4 6 11.9
279.9 6 38.6
199.7 6 28.6
13.42 6 1.31
11.21 6 1.49
15.50 6 2.75
7.78 6 0.65
12.72 6 0.82
21.78 6 2.02
163.5 6 10.6
259.7 6 26.7
177.8 6 13.7
Mice were randomly given phosphoramidon (Phos, 5 mg/kg. i.v.) or thiorphan (Thior, 10 mg/kg, i.v.) before six hourly injections of caerulein (Cae6h, 50 mg/kg, i.p.) or
normal saline. Tissue homogenates were subjected to ELISA assay and normalized with the DNA content of the homogenate and are expressed as picograms per microgram of
DNA (n = 10 mice/group).
*p , 0.05 versus the control,#p , 0.05 versus caerulein-treated mice.
with 10 hourly caerulein injections. One hour after the last caerulein injection, mice were sacrificed and NEP activity and SP ELISA assays were performed
as described in Materials and Methods. A, Pancreas NEP activity. B, Pancreas SP levels. C, Lung NEP activity. D, Lung SP levels. E, Plasma NEP activity.
F, Plasma SP levels. Values are means 6 SEM; n = 10 mice/time point. *p , 0.05 versus control,#p , 0.05 versus caerulein group.
Effect of exogenous NEP on NEP activity and SP levels. Mice received a single dose of normal saline or exogenous NEP (1 mg/kg) along
The Journal of Immunology5435
burns (25, 36). Therefore, the regulatory mechanisms of NEP
activity in vivo might also involve certain proinflammatory mol-
ecules, but they remain to be studied in greater detail.
SP is a major proinflammatory mediator in experimental AP (12,
14, 22). NEP is of critical importance in modulating SP-induced
inflammatory responses, as SP is mainly degraded and inactivated
saline or exogenous NEP (1 mg/kg) along with 10 hourly caerulein injections. One hour after the last caerulein injection, mice were sacrificed and amylase
activity, MPO activity, and water content measurements were performed as described in Materials and Methods. A, Plasma amylase activity. B, Pancreas
MPO activity. C, Pancreas water content. D, Lung MPO activity. E, Lung water content. Values are means 6 SEM; n = 10 mice per time point. *p , 0.05
versus control,#p , 0.05 versus caerulein group.
Effect of exogenous NEP on plasma amylase activity, tissue MPO activity, and tissue water content. Mice received a single dose of normal
Table III. Effect of NEP treatment on expression of cytokines, chemokines, and adhesion molecules
ControlCae10h NEP + Cae10h
In the pancreas
In the lung
3.15 6 0.22
4.08 6 0.27
6.62 6 0.40
2.21 6 0.29
4.11 6 0.24
8.10 6 0.57
26.36 6 1.99
33.96 6 3.11
35.63 6 2.19
7.93 6 0.69*
8.30 6 0.83*
19.02 6 2.08*
5.48 6 0.52*
8.86 6 0.64*
12.77 6 0.96*
40.74 6 2.79*
55.71 6 5.97*
57.99 6 5.80*
5.37 6 0.68*,#
5.30 6 0.58#
8.93 6 1.48#
3.56 6 0.46*,#
6.46 6 0.53*,#
9.35 6 0.91#
27.96 6 2.58#
41.31 6 3.14
42.82 6 4.86
13.52 6 1.12
11.91 6 0.53
10.00 6 0.94
6.87 6 0.44
11.16 6 0.42
21.92 6 1.35
140.4 6 13.9
218.3 6 12.5
199.0 6 16.3
24.75 6 1.88*
13.57 6 2.05
31.82 6 3.99*
11.71 6 0.83*
20.88 6 1.95*
32.98 6 3.83*
173.5 6 16.4
473.5 6 30.8*
235.0 6 20.5
19.03 6 1.69#
12.35 6 0.97
18.07 6 1.94#
9.11 6 0.69#
15.37 6 1.27#
22.57 6 2.17#
135.3 6 16.4
316.5 6 21.9*,#
195.8 6 27.5
Mice were randomly given 10 hourly caerulein (Cae10h; 50 mg/kg, i.p.) injections, and recombinant mouse NEP (1 mg/kg,
i.v.) was given during the second caerulein injection. Tissue homogenates were subjected to ELISA assay and normalized with
the DNA content of the homogenate and are expressed as picograms per microgram of DNA (n = 10 mice/group).
*p , 0.05 versus the control,#p , 0.05 versus caerulein-treated mice.
5436 PROTECTIVE ROLE OF NEP IN CAERULEIN-INDUCED AP
by NEP in vivo (37). In this study, we ascertained the role of NEP
in two opposite ways: inhibition of NEP with phosphoramidon
or thiorphan, or administration of exogenous recombinant mouse
NEP to increase NEP activity. Treatment of mice with exogenous
NEP significantly decreased SP levels in the mice, occurring in
parallel with increased NEP activity. In contrast, NEP inhibitor-
treated mice showed markedly increased SP levels when com-
pared with vehicle-treated mice. Interestingly, exogenous NEP
may be reasonably resilient to metabolism, as markedly elevated
activity in the pancreas, lungs, and plasma was still present 10 h
after treatment. A previous study also suggested that the stability
of exogenous NEP was not compromised in the presence of oxi-
dants or inflammatory fluids (38). Additionally, the inhibitory
effect of phosphoramidon and thiorphan decreased over time,
showing evidence of drug metabolism. In normal healthy mice,
NEP inhibitor treatment still decreased basal NEP activity at the
end of the 8-h treatment protocol. On the contrary, in the AP-
induced mice groups, pretreatment of phorphoramidon or thio-
rphan showed no difference in NEP activity levels with mice that
did not received the treatment. Hence, it appears that the effects of
phosphoramidon and thiorphan were neutralized at a faster rate in
AP-induced mice than in normal, healthy mice.
The present study demonstrates that NEP is protective against
the damaging effects of caerulein-induced AP by modulating
physiological SP levels. Pancreatic edema, increased plasma am-
ylase activity,and infiltrationof neutrophils into the inflamed tissue
are well-known characteristics of caerulein-induced AP (7, 27, 33,
39). In AP, plasma amylase level is markedly increased due to this
enzyme escaping into the blood from damaged pancreatic tissues
(40). Exogenous NEP protected mice against pancreatic damage
and edema, as demonstrated by significantly lowered plasma
amylase activity and tissue water content. On the contrary, mice
pretreated with NEP inhibitors exacerbated pancreatic damage.
These results agreed with previous reports showing the protective
role of NEP in diet-induced hemorrhagic pancreatitis in mice (22).
Our findings were also in line with previous reports showing
potentiated pancreatitis when animals were challenged with ex-
ogenous SP. Exogenous SP elevated pancreatic microvascular per-
meability and interstitial space in caerulein-treated mice, which
contribute to pancreatic microcirculatory dysfunction (41). Ex-
ogenous SP also increased caerulein-stimulated amylase output
(42). Despite this, the effects of exogenous SP administration on
cytokine response in AP-induced animals remain to be explored in
A major feature of AP is the infiltration of neutrophils into the
pancreas. Induction of AP significantly increased MPO activity
in the pancreas, indicating massive neutrophil infiltration. In AP-
induced mice, MPO activity was abolished by exogenous NEP
treatment butfurther increased by NEP inhibitor treatment. Various
molecules are responsible for recruitment of inflammatory cells
and propagation of inflammation. Clinically, the presence of endo-
toxins is linked to more severe cases of AP (43). Exogenous ad-
ministration of endotoxins also strongly propagated inflammation
and caused a much severe form of hemorrhagic AP in mice (44).
However, the absence of detectable endotoxins in the plasma
precludes its role as a proinflammatory mediator in our model of
acute pancreatitis (45). Despite this, a tilt of the balance toward
upregulation of proinflammatory mediators, which include cyto-
kines (IL-1, IL-6, and TNF-a), chemokines (MIP-1a, MIP-2), and
adhesion molecules (ICAM-1, VCAM-1, E-selectin, P-selectin),
was responsible for propagating inflammation in caerulein-
induced AP (46–48). In our study, a significant reduction of
with real-time PCR. A, Inhibition of NEP on NEP mRNA expression. B, Exogenous NEP on NEP mRNA expression. C, Inhibition of NEP on PPTA mRNA
expression. D, Exogenous NEP on PPTA mRNA expression. E, Inhibition of NEP on NK1R expression. F, Exogenous NEP on NK1R expression. Values are
means 6 SEM; n = 8 mice/time point. *p , 0.05 versus control,#p , 0.05 versus caerulein group.
Effect of NEP on mRNA expression of NEP, NK1R, and PPTA in the pancreas. NEP, NK1R, and PPTA mRNA expression was determined
The Journal of Immunology 5437
proinflammatory mediator expression was observed in NEP-
treated mice compared with mice without treatment. SP is able
to upregulate these mediators in leukocytes and pancreatic acinar
cells mainly via activation of NF-kB (49, 50). Thus, NEP
reduces the proinflammatory effects of SP and protects mice
Lung injury is commonly associated with more severe cases of
AP (51). Uncontrolled inflammation in the pancreas elevates
systemic cytokine levels and propagates damage to distant organs,
causing systemic inflammatory response syndrome. In our model
of caerulein-induced AP, the lungs showed similar inflammatory
responses with the pancreas, which were protected by exogenous
NEP. Notably, pretreatment of NEP inhibitors did not aggravate
inflammation in the lung following moderate AP, despite an in-
crease in lung SP levels. It is possible that in our model of
moderate AP, inflammatory responses in the lungs are still in an
early stage, and the effects of SP were still insignificant. None-
theless, the exact role of NEP in the lungs after induction of AP
requires further investigation.
Diminished degradation of SP, in itself, did not appear to cause
inflammation. Mice pretreated with phosphoramidon or thiorphan
without induction of AP raised systemic SP levels but did not cause
changes in tissue MPO activity, water content, and histological
appearance. These observations suggest that agents other than NEP
substrates must first initiate pathological conditions, and then SP
acts as a molecule to promote the progression of inflammation. In
our case, caerulein acted as the initiating agent to cause AP. Ad-
ditionally, slightly increased cytokine/chemokine/adhesion mole-
cule levels were observed in the pancreas and lung after inhibition
of NEP when compared with untreated controls. This is not sur-
prising, as previous studies have shown that the expression of
cytokines and chemokines can be directly upregulated by NK1R
activation via a NF-kB–dependent mechanism (8, 52).
Previous studies have shown disruption of SP–NK1R interac-
tion by a NK1R antagonist, CP96,345, significantly reversed AP-
induced PPTA/NK1R mRNA upregulation in the pancreas (30).
Therefore, it was necessary to test how NEP affects mRNA ex-
pression of PPTA and NK1R after induction of AP. We found that
only NK1R mRNA expression was abolished on NEP-treated, AP-
induced mice, whereas all other treatment groups showed no
significant changes when compared with their respective vehicle-
treated controls. Activation of NK1R may contribute, but not
dictate, the expression of PPTA/NK1R in mice. Furthermore, in
our experiments, exogenous NEP did not completely abolish
physiological SP availability. An in-depth study of SP on the
expression of PPTA/NK1R is necessary to elucidate the mecha-
Preclinical experiments that involve disruption of SP–NK1R
interaction, which include NK1R antagonism and blockade of SP
release from sensory nerve endings, have successfully protected
animals against AP. Results in this study describe targeting NEP
as a novel mechanism to disrupt SP–NK1R interaction during AP,
chiefly via degradation of SP. Administration of exogenous NEP
was previously suggested as a safe and feasible method to protect
animals against various inflammatory disorders (18, 20, 21, 38).
Glucocorticoids, such as dexamethasone, are anti-inflammatory
drugs that were found to increase NEP expression, making it
a potential therapeutic option to target NEP in SP-mediated in-
flammatory responses (53, 54). One major limitation of our study
is that the effects of NEP on other NEP substrates, such as bra-
dykinins and amyloid b, were not adequately addressed. Besides
this, SP can also be degraded by angiotensin-converting enzyme
(16). Future work can be done to address the role of angiotensin-
converting enzyme in SP-mediated responses in AP.
On the basis of this series of experiments, we have concluded
that a high persistent level of SP during AP could be contributed by
a disruption of NEP activity, leading to a detrimental inflammatory
condition that increases and perpetuates pancreatic and lung injury.
Pharmacological inhibition of the SP-degrading enzyme NEP led
to increased availability of SP and exacerbated AP. Conversely,
treatment of mice with exogenous recombinant NEP protects mice
against the detrimental effects of severe AP. Taken together, it is
hoped that the results of these experiments and future studies will
lead to new approaches for the prevention of inflammatory cascade
in patients with AP.
We thank Mei-Leng Shoon (Department of Pharmacology, National Uni-
versity of Singapore) for technical assistance.
The authors have no financial conflicts of interest.
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