J Vet Intern Med 2012;26:875–887
Pathophysiology of Acute Pancreatitis: Potential Application from
Experimental Models and Human Medicine to Dogs
Caroline Mansfield
The cellular events leading to pancreatitis have been studied extensively in experimental models. Understanding the cellular events and inciting causes of the multisystem inflammatory cascades that are activated with this disease is of vital
importance to advance diagnosis and treatment of this condition. Unfortunately, the pathophysiology of pancreatitis in
dogs is not well understood, and extrapolation from experimental and human medicine is necessary. The interplay of the
inflammatory cascades (kinin, complement, cytokine) is extremely complex in both initiating leukocyte migration and perpetuating disease. Recently, nitric oxide (NO) and altered microcirculation of the pancreas have been proposed as major
initiators of inflammation. In addition, the role of the gut is becoming increasingly explored as a cause of oxidative stress
and potentiation of systemic inflammation in pancreatitis.
Key words: Cytokine storm; ROS; Pancreatic necrosis.
A
considerable amount of work has elucidated the
cellular events that initiate pancreatitis and the
resultant inflammatory cascades over the past 3 decades. Most of the investigations have been conducted
on experimental rodent models, with a resultant
extrapolation to naturally occurring disease that may
not always be appropriate.1
Pancreatitis develops when there is excessive activation of trypsin and other pancreatic proteases within
the pancreas that overwhelms both the local safeguards within the acinar cell and antiproteases in the
circulation.2 This initial activation may be caused by
oxidative stress or hypotension, and it is worsened by
low acinar pH and high intracytosolic calcium concentrations.3–7
After initial production of active pancreatic enzymes,
local inflammation ensues. Neutrophil migration then
occurs directly attributable to activation of trypsin and
chymotrypsin, and subsequent production of reactive
oxygen species (ROS) and NO contributes to ongoing
inflammation.8 There is a complex and closely interrelated set of pathways between cytokine production
and this inflammation. Interleukin-8 (IL-8) initiates
neutrophil migration early in the course of the disease,
and upregulates intercellular adhesion molecule-1
(ICAM-1) to promote adhesion of neutrophils to the
endothelial wall.9,10 Major factors that then play a role
in progression of disease include altered pancreatic
microcirculation, ischemia-reperfusion injury, a shift
from acinar cell apoptosis to necrosis, and complex
interaction and stimulation of a variety of inflamma-
Abbreviations:
PSTI
IL
TNF
NF
ROS
NO
NOS
ICAM
RAS
NK
CCK
PLA-2
PAF
pancreatic secretory trypsin inhibitor
Interleukin
tumor necrosis factor
nuclear factor
reactive oxygen species
nitric oxide
nitric oxide synthase
intercellular adhesion molecule
renin angiotensin system
natural killer
cholecystokinin
phospholipase A2
platelet activating factor
tory pathways (complement, kallin-kallikrein, renin
angiotensin system [RAS]).
Unfortunately, despite increased understanding of
the pathophysiology of pancreatitis, a 5–15% mortality
rate is still reported in human medicine.11 This may
reflect the fact that laboratory models tend to focus on
one particular intervention or disease pathway. Consequently, the clinical manifestations and multiple pathways present in naturally occurring disease may not be
taken into account. Despite flaws in translating experimental knowledge to clinical cases, an improved
understanding of the pathophysiology of acute pancreatitis at a cellular level likely will serve to improve
therapeutic interventions in dogs.
Animal Experimental Models
From the Faculty of Veterinary Science, The University of
Melbourne, Werribee, Vic. Australia. All writing was undertaken
at the University of Melbourne, and was not supported by a grant
or other funding body.
Corresponding author: C. Mansfield, Faculty of Veterinary
Science, The University of Melbourne, 250 Princes Highway,
Werribee,Victoria, Australia 3030; e-mail: [email protected].
Submitted December 14, 2011; Revised April 5, 2012;
Accepted April 24, 2012.
Copyright © 2012 by the American College of Veterinary Internal
Medicine
10.1111/j.1939-1676.2012.00949.x
To fully interpret the advances made in understanding the pathophysiology of pancreatitis, it is important
to have an understanding of the experimental models
used and how they may relate to naturally occurring
pancreatitis in dogs. These methods have been well
summarized1 and are reviewed briefly here. The most
commonly applied noninvasive method is administration of a cholecystokinin (CCK) analog (cerulein) IV,
SC, or intraperitoneally. It has been applied to a number of species, including dogs.12 This results in a mild,
self-limiting form of pancreatitis that is useful for
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Mansfield
studying pulmonary pathology, but is probably only
reflective of a proportion of dogs with pancreatitis
seen in veterinary practice. A dietary model based on
feeding a choline-deficient diet with ethionine is simple
and highly reproducible, and causes a severe necrotizing form of pancreatitis.13 This model correlates well
with severe acute pancreatitis seen clinically in dogs
because it results in hypovolemia, hypoxia, pancreatic
necrosis, and acidosis. It is very useful for assessing
interventions that might decrease mortality, but is limited by sex (female) and species (mice) specificity.14
Other noninvasive methods such as ethanol administration, gene knockout, or immune modulation are difficult to reproduce and costly to perform, and seldom
are undertaken unless studying a specific genetic
abnormality or pharmacologic intervention. A recently
developed protocol is administration of high-dose arginine, which results in dose-dependent pancreatic necrosis.15 Long-term administration results in chronic
pancreatitis, which also may be of benefit in canine
medicine as it could allow for evaluation of the insulin-acinar axis, and evaluation of the sentinel acute
pancreatitis event (SAPE) theory which hypothesizes
that chronic pancreatitis results from continued inflammation after an initial acute insult to the pancreas.16
One invasive method of investigating pancreatitis
includes the closed duodenal loop model, which closes
off the pancreatic duct and diverts bile to the jejunum.17 This model induces necrosis rather than inflammation and is associated with a high mortality rate,
often precluding assessment of therapeutic interventions. If the loop is temporarily closed, and trypsin
along with sodium taurocholate is injected into the
duodenum, the pancreatitis is milder and therapeutic
interventions are easier to study.18 It is unknown how
much of a role duodenal reflux plays in causing pancreatitis in dogs, and this model may not be valid for
comparative studies. Perhaps the most promising
methodology for canine disease is combined hypersecretion (injection of CCK) and low-dose infusion of
glycodeoxycholic acid into pancreatic ducts.19 This
method causes widespread necrosis and inflammation
of the pancreas, but poses some technical challenges.
Other invasive methodologies such as antegrade
pancreatic duct perfusion, duct ligation, ischemiaperfusion models, or biliopancreatic duct injections of
taurocholate all produce severe pancreatitis, but are
technically complex.
Pathophysiology of Pancreatitis
Normal Protective Safeguards Against Pancreatitis
The pancreas is able to protect itself from autodigestion in a number of ways. First, some digestive
enzymes are stored in the acinar cells as inert zymogens, and theoretically are only activated after secretion into the lumen of the duodenum.5 Second, within
the acinar cell, the zymogen granules remain physically
separate from the lysosomal granules enclosed in
membrane-bound organelles.20 Lysosomal enzymes are
produced on ribosomes attached to rough endoplasmic
reticulum in the same manner that zymogens are produced, but additionally are glycosolated and phosphorylated as they pass through the Golgi complex.20,21
Studies have shown that these 2 enzyme groups are
kept physically apart throughout all stages of their
production.22 Third, location of pancreatic secretory
trypsin inhibitor (PSTI) within the acinar cells allows
for immediate inhibition of trypsin should it be activated within the acinar cells. PSTI is produced and
stored in the same cellular location as the digestive
enzymes.5,23 Finally, should any activated trypsin be
released into the circulation, larger antiproteases in the
blood theoretically have the capacity to deactivate
some circulating trypsin.
Initiating Cellular Events
Electron microscopy and ultrastructural studies have
confirmed that the earliest event in pancreatitis is activation of trypsinogen to trypsin within the acinar
cell.5,20,24,25 A secretory (or apical) block develops, and
zymogen granules are not secreted normally into the
intestinal lumen for degradation by enterokinase.26
As a result of this apical block, colocalization of
zymogen granules and lysosomal proteases develops,
and trypsinogen is activated to trypsin by lysosomal
proteases, mainly cathepsin B.5,20 This colocalization
theory is depicted in Figure 1, and has become widely
accepted as the main initiating step in pancreatitis.
However, concurrent administration of a cathepsin B
inhibitor significantly decreased development of pancreatitis in 1 rodent study but did not completely prevent it.25 Other mechanisms also may be involved in
trypsin activation because cathepsin B deficient mice
can develop pancreatitis, albeit of lesser severity than
mice without cathepsin B deficiency.27 In addition,
hypotension has been shown to cause trypsin activation before appearance of proteases at the apical surface, suggesting there are other cellular mechanisms
not yet fully elucidated that lead to premature trypsin
activation.4,5 Colocalization may occur because of a
dysfunction of the normally coded separation of the 2
components.28
The role of inappropriate trypsin activation in initiating pancreatitis is exemplified by a mutation in the
cationic trypsinogen gene linked to recurrent pancreatitis in people.29 This mutation may result in failure of
the protective mechanism against activation, and so
prematurely activated trypsin accumulates in the acinar cell.9 A genetic cause of pancreatitis is present in
some canine breeds, such as Miniature Schnauzers, but
the exact genetic abnormalities are yet to be established.30,31
Experimental models have shown that the pH of the
acinar cell is very important in determining the likelihood of lysosomal and zymogen granules colocalizing.6,7,32 The most recent of these studies showed that
low acinar pH alone did not lead to development of
pancreatitis, but it did increase zymogen activation
after cerulein stimulation.6 Trypsinogen activation also
Pathophysiology of Acute Pancreatitis
877
Fig 1. Demonstration of the colocalization theory. In the normal cell on the left, the zymogen granules and lysosomes are manufactured within the Golgi apparatus, but processed and transported to the apex separately. In the abnormal cell on the right, there is an
apical block which allows zymogen granules to fuse with lysosomes. Cathepsin B, a lysosomal protease is then able to activate trypsinogen to trypsin in the acinar cells. Pancreatitis develops when the local safeguard pancreatic secretory trypsin inhibitor (PSTI) is overwhelmed by trypsin, and pancreatic enzymes are then activated within the acinar cell.
appears to require the presence of intracytosolic calcium in some in vitro experiments.33,34 Calcium
located in the zymogen granules may have a protective
function against activation of trypsin, but within the
cytosol it acts in conjunction with CCK as an intracellular messenger for the release of lysosomal proteases.33 In fact, blockade of the calcium signaling
system within cells stops production of trypsinogen
activation peptide.32,34,35
Trypsin and other activated pancreatic enzymes are
likely to be directly damaging to the acinar cell when
production overwhelms local PSTI. In particular,
Phospholipase-A2 (PLA-2) causes hydrolysis of cell
membrane phospholipids, elastase causes degradation
of elastin in blood vessel walls, chymotrypsin activates
xanthine oxidase (thereby perpetuating oxidative damage), and lipase causes fat hydrolysis.36 Early experimental models favor the notion that the spilling over
of activated enzymes into the interstitium is the triggering factor for progression from mild to severe pancreatitis.27 Premature activation of trypsin within the
acinar cells is highly dependent on intracellular pH
and calcium, and can be induced by hypotension and
exacerbated by genetic abnormalities in local safeguards.
Recruitment of Neutrophils and Production of
Reactive Oxygen Species
After this direct damage to the pancreas, inflammation ensues. Inflammation involves vasodilatation of
local blood vessels, increased permeability of local capillaries, clotting of fluid in the interstitial spaces, migration of granulocytes to affected tissue, and swelling of
the tissue cells.37 Neutrophils contain oxidizing agents
such as superoxide (O2!), hydrogen peroxide (H2O2),
and hydroxyl ions (OH!) that are collectively termed
ROS. ROS are directly toxic to cells and further
increase cellular and vascular permeability, as well as
enhancing the expression of inflammatory mediators,
such as eicosanoids released by leukocytes.37 Severity
of pancreatitis was shown to be decreased in mice with
neutrophil depletion.38 However, other studies have
shown that blockade of ROS does not ameliorate
pancreatitis, and thus other factors also must contribute to development of severe disease.39,40
Chemokines are a group of small molecular weight
cytokines that have chemotactic properties, and therefore play a large role in recruiting leukocytes to areas
of injury. In general, they are subdivided into the
CXC and CC subfamilies based on their amino acid
sequence.9 The CXC chemokines that possess the conserved amino acid sequence ELR immediately before
the first cysteine residue at their N terminus are potent
neutrophil attractors, and the best example of this
group is IL-8. Neutrophil migration to the pancreas is
thought to be initiated mainly by IL-8 early in the
course of inflammation.21,41
The adhesion of leukocytes to the endothelial wall
occurs via expression of ICAM-1 and selectins mediated by IL-8 and other chemokines.9,42 ICAM-1 is an
inducible molecule normally only expressed at low levels on endothelial surfaces, but expression has been
shown to be increased in experimental pancreatitis.10
ICAM-1 knockout mice are protected to some extent
against the development of acute pancreatitis and associated organ damage, demonstrating the role of this
molecule in perpetuation of disease.10
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Mansfield
One study recently analyzed the role that elastase
and trypsin play in neutrophil migration in acute
pancreatitis.8 The authors used an in vitro model to
demonstrate that both trypsin and elastase directly upregulate expression of adhesion molecules on white
blood cells and endothelial cells.
Activated neutrophils then follow a chemokine gradient into pancreatic tissue.9 Leukocyte accumulation
occurs initially in the perivascular areas of the pancreas, and then after edema and the resultant change
in permeability they egress to the pancreatic body,
perpetuating the inflammatory pathway within the
pancreas.43
Neutrophils also have been implicated in causing a
shift from apoptosis to necrosis in pancreatitis.3
Necrotic cells release cytosolic contents into the extracellular space and elicit a profound inflammatory
response.21 Apoptotic cells on the other hand are rapidly phagocytized by macrophages and do not elicit an
inflammatory response.44,45 Milder forms of experimental pancreatitis are characterized more by apoptosis than necrosis, and vice versa.46,47 Necrosis is
intricately linked to pancreatic microcirculation, and is
proportional to the degree of oxidative stress.21 Vasoactive mediators also have been implicated in the
development of necrosis (eg, endothelin-1, PLA-2).11
Neutrophil migration into the pancreas is directly stimulated by activated trypsin and chymotrypsin, and
indirectly by upregulation of ICAM-1 by inflammatory
mediators, especially IL-8. Neutrophils themselves
perpetuate an inflammatory state and are involved in
progression from apoptosis to necrosis within the
pancreas.
NO and Oxidative Stress
NO is a small inorganic molecular compound that
has been shown to regulate pancreatic exocrine secretion, promote capillary integrity, inhibit leukocyte
adhesion, and modulate pancreatic microvascular
blood flow.48–50 The biological functions and effects of
NO are complex, and NO can act as both a pro- and
anti-inflammatory agent.51 NOS isoforms are named
after their initial site of location in the vascular endothelium (eNOS), neurons (nNOS), or macrophages
(iNOS). When NO concentration increases within cells
owing to decreased circulation, regional vasodilatation
occurs, but if this persists, mitochondrial respiratory
function is impaired and NO becomes peroxynitrite.50,51 Peroxynitrite is an oxidant that can cause
lipid peroxidation, protein nitration, DNA strand
damage, and cell necrosis.52 Furthermore, NO can
directly produce ROS, thus perpetuating oxidative
stress.48 In experimental pancreatitis, NO has been
shown to be deleterious by increasing oxidative stress
or causing hypotension.53,54 Conversely, NO has been
shown experimentally to have a protective effect in
pancreatitis by increasing blood flow through the
pancreas.55,56
The differences in studies assessing NO may be
explained by the different biological effects of the
different NOS isoforms. In the pancreas, eNOS and
nNOS are constitutively expressed and are calciumdependent enzymes that stimulate low levels of NO
production.48 On the other hand, iNOS is calcium
insensitive and produces large amounts of NO that
circulate throughout the body.48 Studies have shown
that eNOS is downregulated during later stages of
severe pancreatitis and upregulated in mild disease,
whereas iNOS is increased in proportion to disease
severity.49,57–60 Tumor necrosis factor-a (TNF-a) also
decreases expression of eNOS in bovine endothelial
aortic cells, and both nuclear factor kappa-B (NF-jB)
and IL-8 increase expression of iNOS in the lung
and pancreas during experimental pancreatitis in
rodents.59,61–63 NO derived from nNOS is poorly studied in pancreatitis, but could potentially play a role in
amplifying neurogenic inflammation mediated by substance P, as discussed later.64
The exact role of NO in naturally occurring human
pancreatitis still remains unclear.49 One recent study
showed a significant positive correlation between
plasma NO and severity in a group of people with
acute pancreatitis.65 Oxidative stress was greatest in
the group that developed systemic complications. Cigarette smoking and alcohol consumption also may
increase the incidence or severity of acute pancreatitis
by uncoupling eNOS from the NO production, and
causing increased iNOS.48 Despite these assumptions,
therapeutic manipulation of the NO system has had
no proven clinical benefit to date in human medicine
in preventing endoscopic retrograde cholangiopancreatography-induced pancreatitis.48 Additional studies
elucidating the role of NO and the various NOS isoforms are needed in canine pancreatitis. There is experimental evidence that eNOS may be protective in
pancreatitis, whereas iNOS is associated with more
severe pancreatic inflammation.
Pancreatic Microcirculation
The blood flow to, and capillary structure of, the
pancreas are complex and allow integration between
the endocrine and exocrine functions of the pancreas,
which requires a relatively high blood flow to maintain
function.66 In animal models, vasoconstriction within
the pancreas appears to be an early event in acute pancreatitis.67,68 In addition, administration of phenylepinephrine (a potent vasoconstrictor) converts mild,
edematous pancreatitis to a severe, necrotizing form.69
In people, early onset spasm of large pancreatic vessels
has been shown to correlate with poorly perfused areas
of the pancreas, and subsequently with high mortality
rates.70 One experimental study in rats also identified
that lower pancreatic perfusion was associated with
more severe pancreatitis.71
Disturbed pancreatic microcirculation usually is
multifactorial in origin and can occur as a result of
increased vascular permeability resulting from inflammatory cytokines and microthrombi formation resulting from hypercoagulability.66 Increased capillary
permeability leads to edematous changes in the acinar
Pathophysiology of Acute Pancreatitis
cells, and further migration of inflammatory cells
(Fig 2). Necrotizing pancreatitis causes a progressive
reduction in capillaries after acinar cell injury that cannot be reversed by fluid resuscitation.72 The pancreas
is intrinsically susceptible to ischemia, and subclinical
pancreatitis frequently is identified in people with
hypovolemia.73 It remains unknown what degree of
hypovolemia is required to disturb pancreatic microcirculation in dogs.
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Disturbances of pancreatic microcirculation commonly occur in experimental models of pancreatitis,
and contribute to the inflammatory state
Kallin-Kallikrein System
An early and direct action of pancreatic proteases is
to activate the kallikrein system. The pancreas has a
high tissue concentration of kallikrein, generally stored
Fig 2. Pancreatic microcirculation in health and during inflammation. PMN = neutrophil; ICAM = intercellular adhesion molecule.
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Mansfield
as prekallikrein.74 Pancreatic tissue kallikrein releases
kallidin, whereas plasma kallikrein releases bradykinin.
Kinins precipitate edema by vasodilatation of arteries
and increased capillary permeability. They are also
potent activators of nociceptive neurons, can attract
cytotoxic factors into tissue, and enhance production
of prostaglandins.74 There are 2 major kinin receptor
types, with B2 constitutively expressed in tissue and
becoming the major receptor during acute inflammation. The nociceptive and vascular effects of the kinin
system are predominantly mediated by B2 receptors.
Production of kallikreins in acute pancreatitis has been
confirmed in experimental models.74,75 Kinins have a
short biological half-life, and are degraded by kininase
II which is structurally identical to angiotensin-converting enzyme.76 Along similar lines, B2 receptor
antagonists appear experimentally to have beneficial
effects on pancreatic blood flow, necrosis, and survival
in necrotizing models even when given after induction
of disease.77–79 The kinin system may worsen pancreatic edema, and interventions targeting B2 kinin receptors theoretically may be of some benefit in treatment
of disease.
The Complement System
The complement system has been shown to be activated early in people with acute pancreatitis, and to a
greater extent in those with severe disease.80 It remains
unknown to what extent or in what fashion this occurs
in canine acute pancreatitis. Most likely the alternative
complement pathway is initiated in pancreatitis, as
there is an absence of antibodies or microbes as the
primary trigger.36 C5a is one of the most potent mediators of the complement system, and substantially
enhances vascular permeability. Surprisingly, in a
study using mice that either lacked C5a receptors or
did not express C5a, the resultant pancreatitis was
more severe.81 It remains unclear as to why C5a
appeared to have an anti-inflammatory effect in that
study, but it may well be that C5a limits the recruitment of proinflammatory mediators.9,26 In contrast to
this finding, 1 group of researchers found a strong
association between expression of C3a and necrotizing
versus edematous pancreatitis.82 In addition, blockade
of complement receptor-1 led to amelioration of local
pancreatic inflammation and downregulation of the
leukocyte-endothelial interaction. The conflicting conclusions of these studies may be because of the different experimental models used. Alternatively, and more
likely, the complement system may have opposing and
synergistic actions within its cascade. The role of the
complement system in acute pancreatitis is complex
and not fully elucidated in experimental models to
date.
Substance P and the Renin Angiotensin System
Substance P is a peptide produced by nerve endings
that binds to NK-1 receptors. As well as mediating
pain, Substance P likely increases vascular permeabil-
ity, and therefore may play a role in progression of
pancreatitis. A study with a mouse model has demonstrated upregulation of NK-1 receptors and the
amount of Substance P subsequently expressed in
acute pancreatitis.64 Expression of Substance P
appeared to be particularly related to lung injury, and
genetic deletion of NK-1 receptors was protective
against the systemic effects of pancreatitis.64 NO and
Substance P also may interact and synergistically
amplify pain and inflammation.49 No studies to date
have assessed the role of Substance P, or the potential
benefit of treatment with NK-1 receptor antagonists,
in canine pancreatitis.
One area that has been poorly investigated in both
experimental and naturally occurring acute pancreatitis
is the role the RAS may play in the initiation and perpetuation of pancreatic inflammation.43 Angiotensin II
receptors have been identified in the vascular and ductular endothelium of the rodent pancreas.83 Expression
of genes encoding these receptors and metabolites of
the RAS have been shown to be upregulated in acute
pancreatitis, and this may result directly from pancreatic hypoxia.84 The functional effect of this upregulated has not been established, but it is logical that
local vasoconstriction would ensue. Substance P inhibition theoretically could decrease pain and lung-associated injury, and the pancreatic RAS may stimulate
vasoconstriction in acute pancreatitis.
Cytokine “Storm”
Before, during, and after inflammatory cells have
appeared within the pancreas many cytokines and
chemokines are activated. This is a highly complex
phenomenon, and has been referred to as “cytokine
storm”, with many cytokines contributing to the
inflammatory state.42,85,86 Cytokines are small proteins
produced in response to stimuli, and act to up- or
downregulate various aspects of the inflammatory
process. Many cytokines are produced by T cells to
either drive or facilitate a Th1 or Th2 immune
response. Th1 responses mediate cellular immunity and
activate neutrophils, macrophages, and NK cells. Th2
responses mediate humoral immunity by B cell activation or recruitment of eosinophils. Each cytokine has
the capacity to act synergistically with others, and
there is a large overlap in their functions (Fig 3).
The initiating step of this process in pancreatitis, as
in many other systemic inflammatory diseases, appears
to be the activation of NF-jB.87,88 NF-jB is a transcription factor that modulates the expression of most
cytokines. High NF-jB expression has been identified
in peripheral blood mononuclear cells from people
with acute pancreatitis, and correlates with development of systemic complications.89 NF-jB also stimulates production of adhesion molecules and iNOS.62
In 1 rodent model, cytokine activity within the pancreas was determined by tissue mRNA and RT-PCR,
and TNF-a and IL-1b were the first cytokines to
appear.90 The amplitude of cytokine expression correlated with the severity of pancreatic inflammation.
Pathophysiology of Acute Pancreatitis
Ac va on of inflammatory cells
Pro-inflammatory
mediators:
TNF-α; IL-1,-2,-6; NO; ROS;
AA metabolites
An inflammatory
mediators:
Chemokines:
IL-8; MCP-1; RANTES
IL-10,-4,-1ra
Systemic
inflamma on
Fig 3. Demonstration of the activation of the cytokine storm in
acute pancreatitis. Systemic inflammation results from the cytotoxic effects when the proinflammatory mediators (red) overwhelm the anti-inflammatory mediators (blue). TNF, tumor
necrosis factor; IL-, interleukin; ra, receptor antagonist; NO,
nitric oxide; ROS, reactive oxygen species or superoxide; AA,
arachidonic acid; MCP-1, monocyte chemoattractant protein-1;
RANTES, regulated on activation, normal T cell expressed and
secreted. Solid lines represent stimulatory pathways, whereas dotted lines represent inhibitory pathways.
Pancreatic acinar cells have been shown to be capable of producing TNF-a.91,92 TNF-a is synergistic with
many other inflammatory cytokines, and also promotes cell adhesion (via ICAM-1) and programmed
cell death.42 IL-1 is a proinflammatory cytokine that is
synergistic with TNF-a. The production of IL-1
requires IL-1b and 2 IL-1 receptors that have similarities to toll-like receptors. This production is mediated
by a group of enzymes called caspases, which also
cause the production of other inflammatory cytokines,
such as IL-8. The caspase family (previously known as
interleukin-1 converting enzyme) appears to favor
necrosis over apoptosis compared with TNF-amediated inflammation in acinar cells, and worsens the
severity of pancreatitis.93–95
IL-1b is a potent facilitator of neutrophil migration
and is strongly associated with the development of systemic inflammation.96 In addition, IL-1b activates the
transcription of ICAM-1 and NF-jB, further perpetuating the inflammatory cascade. Early activation of
this cytokine has been demonstrated in experimental
models of acute pancreatitis.90,97,98 The effects of IL1b are counteracted by an IL-1 receptor antagonist
(IL-1ra). In people with acute pancreatitis, an increase
in IL-1b to IL-1ra ratio was highly predictive of pulmonary failure.86 Furthermore, in an experimental
model, IL-1 knockout mice did not develop pancreatitis, emphasizing the role this cytokine plays in the
early initiation and progression of acute pancreatitis.99
IL-6 is a proinflammatory cytokine produced by a
variety of mononuclear cells, endothelial cells, and
fibroblasts. It is produced in response to stimulation
881
by TNF-a and IL-1b, and is increased early in the
course of experimental pancreatitis.42,90 IL-6 is a relatively easy cytokine to measure and has been negatively correlated with prognosis in naturally occurring
acute pancreatitis in humans.86,100–103
IL-8 is a proinflammatory chemokine closely
involved in neutrophil actions, both as an inciter and
product of the Th1 profile. Similar to IL-6, the concentration of IL-8 has been correlated with the severity of
naturally occurring pancreatitis in people.100,103,104
Platelet activating factor (PAF) is a cytokine produced
predominantly by neutrophils that has been extensively
studied in experimental models. It appears to be particularly associated with lung injury, which is one of
the major causes of early (<2 weeks) mortality in
people with acute pancreatitis.3,105,106 Blockade of PAF
has led to reduction in lung-associated complications in
people with pancreatitis in some studies, but overall,
did not improve mortality or other organ failure.107–109
In experimental studies, IL-10 has been assessed
because of its anti-inflammatory effect, which favors a
shift to a Th2 cytokine profile and downregulates
NF-jB transcription. Some initially promising results
demonstrated that IL-10 ameliorated the severity of
pancreatitis in experimental models.110,111 Pretreatment
of rats with IL-10 decreased iNOS concentrations, and
increased TGF-b1, which correlated with an increase
in the rate of apoptosis as compared with necrosis.112
In addition, IL-10 knockout mice were shown to
develop a more severe form of pancreatitis.113 These
findings, along with demonstration that serum IL-10
was increased in people with severe pancreatitis within
the first 2 days of disease, but subsequently decreased,
suggested a potential treatment avenue.114 Unfortunately, IL-10 therapy has had limited success in treatment of acute pancreatitis in humans.43
As knowledge in this area continues to increase,
many other cytokines (eg, IL- 2, -4, -11, and -18) are
also being evaluated in experimental pancreatitis.86,94
Regulated on activation, normal T cells expressed and
secreted (RANTES) is now known as CC chemokine
ligand 5 (CCL5), and is produced by circulating T cells
under stimulation by TNF-a and IL-1.42,115 It is
responsible for protection against viruses, and is a
potent chemoattractant for recruiting leukocytes.42 The
role of CCL5/RANTES or other potent chemoattractants, such as monocyte chemoattractant proteins
(MCP)-9 in canine pancreatitis remains unknown.
Whether these inflammatory mediators become clinically important in canine pancreatitis from a therapeutic or etiologic point of view remains to be seen. There
are a large number of complex and interdependent
cytokines produced in acute pancreatitis. This suggests
that intervention targeting a single cytokine is unlikely
to be effective in modulating disease.
Perpetuation of Disease
With resultant hypovolemia from vomiting, diarrhea,
and anorexia, the splanchnic circulation is sacrificed
early and may contribute greatly to the perpetuation of
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the inflammatory state in pancreatitis.3 Hepatosplanchnic circulation accounts for 20–30% of cardiac output.116 In acute pancreatitis, substantial third space
fluid loss occurs, and splanchnic circulation subsequently decreases as part of hypovolemic shock. The
body adjusts by increasing oxygen extraction from systemic circulation up to 90%, whereas it is thought that
prolonged oxygen extraction >70% can lead to regional ischemia.117 Ischemia of the intestine increases permeability and mucosal acidosis, which increase the rate
of apoptosis of enterocytes and decrease nutrient
transport of intestinal epithelial cells.118 In addition,
increased bowel permeability ensues and leads to further cytokine release and skeletal muscle lysis.119
Two proposed models suggest how the intestine may
contribute to the inflammatory state, although in reality it is likely to be a combination of both.120 In the
“Gut Starter” model, neutrophils are primed during
reperfusion of the ischemic intestine, and then are provoked by exposure to endotoxin in systemic circulation.121 This is mediated by PLA-2 in the mesenteric
blood system.122 The primed neutrophils are then
potent mediators of distant organ dysfunction. In the
“Gut Motor” model, disruption of the intestinal barrier is thought to lead to translocation of bacteria and
inflammatory cells into the portal venous and lymphatic systems, which activates downstream immune cells
in the absence of infective foci.123
One experimental pancreatitis study in pigs showed
that decreased intestinal blood flow caused mucosal
acidosis.124 In people with severe acute pancreatitis,
low mucosal pH has been shown to be closely related
to development of complications and death.125 Xanthine oxidase also may be produced on reperfusion of
ischemic intestine, leading to production of free radicals that perpetuate the inflammatory process.118
The intestinal barrier is a functional unit that consists of intestinal microflora, appropriate gut motility,
digestive enzymes, mucus layer, epithelial layer, endothelial layer, mucosal-associated lymphatic tissue, and
the gut-liver axis.119,120 The epithelial layer is thought
to be the most important part of the intestinal barrier
because it is a single cell layer that is very prone to
disturbances in microcirculation.120 In people and cats,
the oxygen counter current exchange system and anatomical position of the villi circulation have been
shown to predispose to hypoperfusion during times of
decreased splanchnic circulation.118,126 In intestinal
villi, the artery and vein run in parallel, but opposite
directions, with a dense capillary network close to the
top of the villus. This anatomy results in a decreasing
tissue PO2 gradient from the base up to the tip of the
villus.116 The change in PO2 also may be because of
the higher metabolic activity of cells located in the tip.
Regardless of the mechanism, this low PO2 makes the
villi tips susceptible to tissue hypoxia if vasoconstriction occurs. In dogs, a similar vascular anatomy of the
villus is present, with a single arteriole rising
unbranched to the tip of the villus.127 Dogs may be
exquisitely sensitive to villus hypoxia because drainage
from the capillary network to the veins occurs closer
to the tip of the villus than in other species.127 The
intestine is susceptible to ischemia during acute pancreatitis, and upon reperfusion may be the initiator or
perpetuator of distant inflammation by a variety of
mechanisms.
Pathophysiology in Experimental Models Related to
Potential Etiologies in Dogs
The oxidative theory of pancreatitis was first proposed in human medicine in the early 1980s.128
According to this theory, the pancreas is exposed to
oxidative stress either through the systemic circulation,
or via reflux of bile containing reactive metabolites
into the pancreatic duct. In people, the most common
oxidative stressors include alcohol and high-fat diets.16
However, oxidative stress currently is thought to
accompany but not directly cause pancreatitis without
other factors also being present.16
Experimental evidence also suggests that lowprotein, high-fat diets induce pancreatitis in dogs, and
that high-fat diets in dogs cause more severe pancreatitis than fat-restricted diets.129,130 These studies, however, did not assess whether pancreatic necrosis or
inflammation was present, rather the volume of pancreatic secretion and the protein concentration of those
secretions was assessed. Overweight dogs are considered at greater risk of pancreatitis, and this risk may
be associated with abnormal dietary intake or may
indicate a general predisposition to inflammation.131,132
A chronic inflammatory state is associated with adipose
tissue and adipokines in people, and a similar association likely also occurs in dogs.133,134 In 1 retrospective
survey, dogs with recent ingestion of unusual food
items and garbage ingestion showed increased risk of
developing pancreatitis, rather than dogs that appeared
to have a higher intake of treats and snacks.132 This
study suggested, but could not prove, that inappropriate food rather than the fat and protein content of
food may be most important in the development of
pancreatitis.
Concurrent diseases, particularly diabetes mellitus, hypothyroidism, and hyperadrenocorticism, are
reported commonly in dogs with pancreatitis.131,132,135
Some possible connections between diabetes mellitus
and pancreatitis include subclinical pancreatitis causing
progressive islet cell destruction or autoantibodies
directed against insulin-secreting cells promoting generalized pancreatic inflammation. Acidosis within the
pancreas in diabetic ketoacidosis (DKA) may enhance
or initiate trypsin activation, rather than DKA being
caused by pancreatitis.6 Endocrinopathies are associated with changes in serum lipid concentrations, and
the association between hyperlipidemia and pancreatitis cannot be discounted.136,137 Alterations in bile composition with varying lipid status may directly lead to
toxic changes in pancreatic acinar cells by changing
cellular metabolism.16
Acute pancreatitis also may directly result from
hypoxia and ductal hypertension, by an effect on the
pancreatic microcirculation.4,73,138 This theoretically
Pathophysiology of Acute Pancreatitis
may account for the high incidence of pancreatitis
reported after abdominal surgery, especially adrenalectomy.132,139 Despite the experimental association
between premature trypsin activation and hypercalcemia, common conditions such as lymphoma that cause
hypercalcemia are not reported to cause pancreatitis in
dogs. The lack of reported association between pancreatitis and hypercalcemia in dogs may be because of the
fact that the increase in calcium is more gradual than
that seen in people, where hypercalcemia-associated
pancreatitis most often is seen with cardiopulmonary
bypass.138,140 It also is possible that the pancreatitis is
subclinical and escapes diagnosis in these dogs.
Development of Complications
Acute renal failure may develop secondary to hypovolemia, ischemia, intravascular coagulopathy, and
direct inflammation resulting from peritonitis.141 NFjB activation also can cause aggregation of activated
neutrophils in the glomeruli.89 Endotoxin may promote renal vasoconstriction contributing to renal failure attributable to high affinity binding on the renal
artery.142 Other systemic complications include disseminated intravascular coagulation and cardiac arrhythmias, all mediated by the many systemic inflammatory
cascades initiated by acute pancreatitis.143,144
Acute lung injury is most closely linked to PAF,
although PLA-2, TNF-a, and IL-1b may also play a
role.145 The histologic changes in lungs undergoing
acute injury during experimental pancreatitis include
neutrophilic infiltration, damage of endothelial cells,
interstitial edema, and intra-alveolar hemorrhage.146 In
addition, there may be changes in pulmonary surfactant
or apoptosis of type II pneumocytes.146 Acute lung
injury is considered one of the major early complications in people, and is associated with high morbidity.3
The actual incidence of lung injury in dogs with acute
pancreatitis remains unknown, but a study assessing
clinical severity did identify alterations in respiratory
function as significantly affecting clinical outcome.147
Late onset complications include chronic relapsing
pancreatitis and subsequent development of exocrine
pancreatic insufficiency, both of which have been
described in dogs.148,149 Recently in people, subclinical
exocrine pancreatic insufficiency has been demonstrated after a bout of severe acute pancreatitis.150,151
Subclinical exocrine pancreatic insufficiency is difficult
to diagnose in dogs, but is highly probable.152
Acute fluid collections are defined in the human literature as fluid pockets within the pancreatic parenchyma that develop within the first 6 weeks after a
bout of acute pancreatitis.153 A pseudocyst on the
other hand, develops at least 6 weeks after an episode, does not contain an epithelial lining and its
contents are composed of amylase-rich pancreatic
secretion, generally occurring in milder cases of acute
pancreatitis.153 Another late onset (3–4 weeks) local
complication is a hypovascular area containing necrotic tissue that in people may become a nidus for
infection.153
883
The veterinary literature suggests development of
fluid in the canine pancreas is invariably acute in onset
and sterile in nature.154–160 This is much different from
the clinical situation in people, where infected necrosis
is one of the major determinants of mortality. These
findings suggest that the local complications within the
pancreas of the dog instead should more consistently
be called acute fluid collections, unless they are confirmed to be infected. The reported mortality rates for
dogs with pancreatic abscesses are high (>50%),
but this may be reflective of fact that all have been
treated surgically with subsequent complications.154–160
Current recommendations in human gastroenterology
are to not surgically debride sterile fluid collections,
and if infection is documented treatment with antimicrobials should be employed for as long as possible
before surgical debridement.107
Conclusion
Acute pancreatitis has a complex pathogenesis that
involves multiple inflammatory pathways. The pancreas itself is exquisitely sensitive to circulatory and
ischemic events. Even if these events are not the cause
of the pancreatic inflammation, their development during the ensuing clinical dehydration has the potential
to worsen disease. In recent times, the roles of the
kinin-kallikrein system, substance P, pancreatic microcirculation, and perpetuation of disease by the intestinal tract all have received substantial attention.
Intervention in these areas appears to have the most
promise in attenuating disease severity as compared
with treatments that counteract single cytokines.
Acknowledgment
Kate Patterson from MediPics and Prose provided
medical illustrations
Conflict of interest statement: Dr Mansfield is on the
Australian Emesis advisory panel for Pfizer, Australia.
Pfizer had no role in the writing of this manuscript.
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