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JOURNAL OF SURGICAL RESEARCH 74, 3±7 (1998)
ARTICLE NO. JR975174
TNF but Not IL-1 Decreases Pancreatic Acinar Cell Survival without
Affecting Exocrine Function: A Study in the Perfused Human Pancreas
Woody Denham, M.D., Jun Yang, M.D., Greg Fink, M.D., Daphne Denham, M.D., Gay Carter,
Victor Bowers, M.D., and James Norman, M.D.1
Department of Surgery and the Pancreas Study Group, University of South Florida, Tampa, Florida 33612
Presented at the Annual Symposium of the Association of Veterans Administration Surgeons, Louisville, Kentucky, May 5±7, 1997
INTRODUCTION
Substantial quantities of interleukin-1b (IL-1b) and
tumor necrosis factor-a (TNF-a) are produced within The pathophysiology of acute pancreatitis (AP), a
the pancreatic parenchyma during acute pancreatitis. noninfectious in¯ammation of the pancreas, remains
Recent evidence suggests that IL-1b and TNF-a propa- poorly understood. Recent investigations implicate
gate acute pancreatitis and intensify the resulting pan- the proin¯ammatory cytokines interleukin-1b (IL-
creatic acinar cell death. This study examines the direct 1b) and tumor necrosis factor-a (TNF-a) as central,
effect of IL-1b and TNF-a on pancreatic acinar cells. detrimental mediators in the progression of AP. IL-
Human pancreata (n  6), harvested during organ pro- 1b and TNF-a are elevated in the serum of humans
curement, were perfused ex vivo through the splenic and experimental animals with AP regardless of the
artery using a sterile, oxygenated colloid solution. Each model studied [1±6]. Pancreatic parenchymal levelspancreas was perfused with either recombinant human
of IL-1b and TNF-a are undetectable in normal ani-IL-1b or TNF-a for 2 h and subsequently with the chole-
mals; however, induction of AP results in markedlycystokinin analogue caerulein (positive control). Ve-
increased concentrations of both cytokines within thenous ef¯uent was collected continuously and amylase
pancreas [4±7]. The serum and tissue concentrationsand lipase were determined at 15-min intervals. Pan-
of IL-1b and TNF-a are typically predictive of diseasecreatic histology was graded at baseline and following
severity and correspond with subsequent morbiditycytokine and caerulein perfusion. To examine the long-
and mortality [2, 4±7].term effects of these cytokines on acinar cell viability,
Evidence for the importance of IL-1b and TNF-a pro-additional in vitro studies utilized the AR42J acinar cell
duction has been largely demonstrated by studies uti-line which was exposed to either IL-1b or TNF-a with
lizing pharmacologic antagonism. When either IL-1bsurvival determined daily by MTT assay. Perfusion of
or TNF-a is inhibited using agents such as receptorthe human pancreas with either IL-1b or TNF-a did not
antagonists or binding proteins, the severity of pancre-alter amylase, lipase, or histology. Caerulein did induce
pancreatitis as measured by increased amylase, lipase, atitis is attenuated and mortality is decreased [8±11].
and pancreatic histology. Survival of pancreatic acinar Inhibition of either cytokine markedly decreases pan-
cells decreased when they were incubated with TNF-a creatic necrosis and acinar cell injury in animal models.
but not IL-1b. Although present in large amounts These improvements are seen with prophylactic or
within the pancreas during acute pancreatitis, IL-1b therapeutic blockade of IL-1b or TNF-a. Further proof
and TNF-a have no direct effect on acinar cell viability of the important role for IL-1b and TNF-a has been
or exocrine function acutely nor do they induce pancre- demonstrated in transgenic animals which are devoid
atitis. When present for more than 24 h, however, TNF- of active receptors for either IL-1 (p80) or TNF (p55).
a but not IL-1b has a dramatic effect on acinar cell When AP is induced in these receptor-knockout ani-
survival. q 1998 Academic Press mals, pancreatic acinar cell injury is lessened, resulting
in decreased severity and mortality compared to wild-
type animals with normal receptors [12].
The central role that active IL-1b and TNF-a play
1 Supported by a Veteran's Administration Merit Review Grant.
in the propagation and progression of AP is clear, and
studies have demonstrated that these cytokines are re-To whom correspondence should be addressed at University of South
quired for maximal progression of this disease [11, 13,Florida, Department of Surgery, Box 1289, Tampa General Hospital,
Tampa, FL 33601. Fax: (813) 251-7396. 14]. When either cytokine is inhibited, pancreatic aci-
3 0022-4804/98 $25.00
Copyright q 1998 by Academic Press
All rights of reproduction in any form reserved.
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4 JOURNAL OF SURGICAL RESEARCH: VOL. 74, NO. 1, JANUARY 1998
nar cell injury is lessened which implies that both IL-
1b and TNF-a may be responsible for the destruction
of acinar cells during the progression of AP. This study
was undertaken to determine the direct effect(s) of
short- and long-term pancreatic acinar cell exposure to
IL-1b and TNF-a.
METHODS
Human Perfused Pancreas
All studies using human tissue were performed with prior ap-
proval of the Institutional Review Board at the University of South
Florida. Human pancreata were harvested during organ procure-
ment from six cadaveric donors following brain death due to sub-
arachnoid hemorrhage (n  4) or trauma (n  2). There was no
evidence of previous pancreatic disease, and no donor had clinical
evidence of pancreatitis as determined by serum amylase and li-
pase prior to harvest.
Following the removal of other organs for transplantation, the
pancreas was harvested as previously described [15]. Brie¯y, the
pancreas was mobilized from the retroperitoneum in continuity
with the spleen from lateral to medial. The splenic artery and vein
were identi®ed along the posterior surface of the pancreas, and
the splenic artery was cannulated with a 14-gauge catheter. The
pancreas was perfused in situ with cold lactated Ringer's solution.
An 8Fr feeding tube with multiple side holes was placed in the
splenic vein and secured. The pancreas was then transported to
the laboratory at 47C.
On arrival to the laboratory, the human pancreas was immedi-
ately placed on an ex vivo perfusion circuit with a peristaltic pump
(Brinkmann Instruments, Roxbury, NY) and constant in-line mon-
itoring to ensure that the perfusion pressure was never greater
than 30 cm of water. The perfusion media was a Kreb's-Ringer's
bicarbonate buffer containing 70 mg/dl glucose, 3% type-speci®c
human fresh-frozen plasma (Southwest Regional Blood Bank,
Tampa, FL), and heparin (100 U/dl). The medium was warmed to
377C and oxygenated by passing through a diffusion oxygenator
perfused with 95% O2 and 5% CO2.
Baseline values were achieved by perfusing each pancreas with FIG. 1. Amylase (A) and lipase (B) determined in the venous
media alone for 2 h. Following this equilibration period, recombi- ef¯uent of human pancreata (n  3) perfused with IL-1b. Baseline
nant human IL-1b (20 ng/mg, National Cancer Institute, Freder- values (perfusate only) for both enzymes did not increase when IL-
ick, MD) or human TNF-a (20 ng/mg, Endogen, Cambridge, MA) 1b was added. Perfusion with the cholecystokinin analogue caerulein
was added to the perfusate. Each pancreas was then perfused with (positive control) resulted in a marked increase in amylase and lipase
either IL-1b (n  3) or TNF-a (n  3) for 2 h. The cholecystokinin (*P › 0.05 vs baseline and IL-1b).
analogue caerulein (50 mg/kg) was then added to fresh perfusate
for an additional 2-h perfusion serving as a positive control. Ve-
well plates in Kaighn's modi®ed F-12K HAM media (Sigma, St.nous ef¯uent was collected continuously in 15-min aliquots during
Louis, MO) supplemented with 20% fetal calf serum and allowedthe entirety of pancreatic perfusion and stored at 0807C until
to incubate overnight. Recombinantrat IL-1b or TNF-a (Endogen,analyzed.
Cambridge, MA) was then added to each plate (200 ng/ml). Pancre-
atic acinar cell viability was determined at 0, 24, 48, 72, and 96 h
Determination of Pancreatic Exocrine Function and using a nonradioactive cell proliferation assay (Promega, Madison,
Acinar Cell Injury WI). Brie¯y, a dye solution containing 3-[4,5-dimethylthiazol-2-yl]-
2,3-diphenyltetrazolium bromide (MTT) was added to each well for
Amylase and lipase. Amylase and lipase levels were determined 4 h. Using a plate reader (Dynatech Laboratories, Chantilly, VA),
in the venous ef¯uent using a Kodak Ektachem 700 automated ana- the absorbance was determined at 570 nm, and the number of viable
lyzer (Kodak, Rochester, NY). All samples were run in triplicate and cells was calculated from a standard curve. All samples were run in
averaged. triplicate and averaged.
Pancreatic histology. Pancreatic tissue was collected and ®xed in Statistical methods. Results are expressed as means{ SEM. Sta-
10% buffered formalin at the beginning of the perfusion and following tistical analysis was performed using the EPISTAT statistical pro-
exposure to IL-1b or TNF-a, and caerulein (three samples per per- gram (Epistat Services, Richardson, TX) applying unpaired two-
fused pancreas). Fixed pancreatic tissues were embedded in paraf®n tailed Student's t test with signi®cance being assigned to P values
and stained with hematoxylin and eosin. Blind histologic grading of › 0.05 unless otherwise stated.
pancreatic edema, necrosis, and vacuolization (range 0 to 4) was
RESULTSperformed on 10 high-powered ®elds from each sample as previously
described [9]. Human Perfused Pancreas
In vitro acinar cell viability. The rat pancreatic acinar cell line
Acinar cell function. To examine acinar cell func-AR42J was utilized to determine the long-term effect of acinar cell
exposure to IL-1b or TNF-a. AR42J cells (104/ml) were seeded in 96- tion, amylase and lipase were determined in the ve-
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5DENHAM ET AL.: IL-1 AND TNF IN PERFUSED HUMAN PANCREAS
TABLE 1
Blind Histologic Scoring of Human Pancreata
Baseline IL-1b TNF-a Caerulein
Edema 0 0.1 0.1 3*
Necrosis 0 0.1 0.1 0.3**
Vacuolization 0 0 0 0.5*
Total 0 0.1 0.1 3.8*
Note. Histologic sections of each pancreas were graded in a blinded
fashion for edema, necrosis, and vacuolization. Normal tissues were
assigned a value of 0 while maximal severity for each parameter was
assigned a value of 4. Values represent the means { SEM for 10
®elds from each pancreatic specimen. *P › 0.05 vs baseline, IL-1b,
and TNF-a. **P › 0.05 vs baseline.
ine pancreatic acinar cells for injury (Table 1). Normal
pancreata were assigned a score of zero for edema, ne-
crosis, and vacuolization. Addition of IL-1b or TNF-a
to the perfusate did not alter these three parameters (P
 ns). When caerulein was added as a positive control,
edema and vacuolization worsened (P › 0.05 vs base-
line). Additionally, there was more necrosis with caer-
ulein perfusion; however, it was not statistically sig-
ni®cant compared to perfusion with IL-1b or TNF-a.
Acinar Cell Viability in Vitro
The viability of the pancreatic acinar cell line AR42J
was examined during incubation with media alone, IL-
1b, or TNF-a (Fig. 3). IL-1b had little or no effect on
acinar cell viability as determined by MTT assay (P
 ns vs media alone). Exposure to TNF-a resulted inFIG. 2. Human pancreata (n  3) perfused with TNF-a. Amy-
decreased viability at 48, 72, and 96 h (P › 0.05 vslase (A) and lipase (B) were determined at 15-min intervals from
media).the venous ef¯uent. Perfusion with TNF-a did not induce acinar
cell injury as measured by these two enzymes. Perfusion with the
cholecystokinin analogue caerulein (positive control) resulted in a
signi®cant increase in amylase and lipase (*P › 0.05 vs baseline
and TNF-a).
nous ef¯uent collected from the perfused pancreas.
Figure 1 shows amylase (A) and lipase (B) from pan-
creata perfused with IL-1b. The enzyme levels mea-
sured during the equilibration period (perfusate
alone) did not increase when IL-1b was added (P 
ns). Addition of caerulein to fresh perfusate resulted
in a dramatic increase in venous amylase and lipase
compared to baseline or perfusion with IL-1b (both
P › 0.05). Similarly, perfusing the pancreas with
TNF-a (Fig. 2) did not result in an increase in venous
amylase or lipase (P  ns vs baseline). Again, perfu-
sion with caerulein caused a multifold increase in
the production of amylase and lipase compared to
perfusion with media with or without TNF-a (both P
FIG. 3. Acinar cell viability in vitro. Exposure of the pancreatic› 0.05).
acinar cell line AR42J to IL-1b for 4 days did not affect viability.
Pancreatic histology. Multiple pancreatic sections Incubation with TNF-a resulted in decreased viability at 48, 72, and
96 h (*P › 0.05 vs media alone).were graded (range 0 to 4) by light microscopy to exam-
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6 JOURNAL OF SURGICAL RESEARCH: VOL. 74, NO. 1, JANUARY 1998
DISCUSSION sure [20] but have not yet been examined in acinar
cells.
The central role of IL-1b and TNF-a during the pro-
gression of AP has recently been con®rmed in a number ACKNOWLEDGMENTS
of studies. Regardless of the experimental model of pan-
creatitis, IL-1b and TNF-a are increased in the serum We are indebted to F. Charles Brunicardi, M.D. (Department of
Surgery, Baylor College of Medicine, Houston, TX) for his work inand pancreatic parenchyma [3±7]. When either cytokine
developing the perfused human pancreas, and we are grateful foris blocked, pancreatic acinar cell injury is lessened as
his time and effort in sharing this model with our laboratory.measured by attenuated serum amylase and lipase [8±
11]. Additionally, pancreatic necrosis, edema, in¯amma-
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