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Acute Pancreatitis-Induced Enzyme Release and Necrosis Are Attenuated by IL-1 Antagonism through an Indirect Mechanism
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
67, 94–97 (1997)
JR964935
Acute Pancreatitis-Induced Enzyme Release and Necrosis Are Attenuated by IL-1 Antagonism through an Indirect Mechanism GREGORY FINK, M.D., JUN YANG, M.D., GAY CARTER,
AND
JAMES NORMAN, M.D.1
Department of Surgery and the Pancreas Study Group, University of South Florida, Tampa, Florida Presented at the Annual Symposium of the Association of Veterans Administration Surgeons, Detroit, Michigan, April 28–30, 1996
in excess of 100 ng/ml during peak periods of inflammation during pancreatitis [5, 6]. Subsequent experiments have suggested that intrapancreatic IL-1b originates from within infiltrating leukocytes with no evidence available at this time to suggest production by pancreatic acinar or ductal cells [7]. Although IL-1b production correlates with pancreatic destruction, it is not felt to be just a marker of the severity of the disease, but rather an important intermediary in the progression of acute pancreatitis. Evidence for this influential role is provided by several studies demonstrating decreased mortality in lethal models of pancreatitis when IL-1b is either pharmacologically or genetically rendered inactive [8–10]. The mechanism by which IL-1b blockade improves the severity and mortality of experimental pancreatitis has not been completely delineated. We have shown that acinar cells constitutively express mRNA for both IL-1 type I and type II receptors and possess the receptors on their cell surface (unpublished data). Despite these findings, it is not known whether IL-1 serves a physiologic purpose within the pancreas at rest or during acute pancreatitis. This study was undertaken to test the hypothesis that IL-1b contributes to the severity of pancreatitis through direct effects on acinar exocrine function and detrimental effects on acinar viability.
Interleukin-1b (IL-1) is a proinflammatory cytokine which is produced within the pancreas during acute pancreatitis reaching levels which are toxic to many cell types. Since antagonism of this cytokine provides dramatic survival benefits during lethal pancreatitis, we hypothesized that IL-1 had direct secretagogue and cytolytic effects within the pancreas. The effect of IL1 on pancreatic exocrine function and tissue viability was assessed in vivo by blockade of IL-1 with varying doses of IL-1 receptor antagonist (IL-1ra) prior to the induction of either moderate (caerulein-induced) or severe (choline deficient diet-induced) necrotizing pancreatitis. Subsequent in vitro studies were conducted to determine the direct effect of IL-1 on dispersed rat acini prepared through collagenase digestion. Amylase release was measured after a 30-min incubation with varying doses of recombinant IL-1b. Viability was determined in the presence of IL-1 via trypan blue exclusion at multiple time points. Blockade of the IL-1 receptor decreased pancreatic amylase release and tissue necrosis in both models of pancreatitis in a dose-dependent fashion (1.0 mg/kg, P Å NS; 10 mg/kg, P õ 0.05; 100 mg/kg, P õ 0.05). Despite these in vivo findings, the addition of IL-1 to acini in vitro had no effect on exocrine function and failed to decrease acini viability (both, P Å NS). Pancreatic amylase release and tissue necrosis are significantly attenuated during experimental pancreatitis by IL-1 antagonism. These changes do not appear to be due to the direct action of IL-1 on pancreatic acini and are likely due to more complex interactions between acini and cytokine-producing leukocytes. q 1997 Academic Press
METHODS In vivo pancreatitis: Animal model. Animal studies were performed at an AAALAC accredited facility in accordance with the Department of Laboratory Animal Medicine at the University of South Florida. Two models of pancreatitis were utilized. A well-established model of acute moderately necrotizing pancreatitis was employed in 72 adult male NIH Swiss mice (32 { 0.3 g) by the intraperitoneal (ip) injection of caerulein (50 mg/kg/hr 1 4), a cholecystokinin analogue [11, 12]. Control mice received ip saline at the same time intervals. Animals were sacrificed at 0 and 8 hr after pancreatitis induction. In the second model, severe necrotizing pancreatitis was induced by feeding a choline-deficient ethionine-supplemented diet to young female NIH Swiss mice (15.3 { 0.2 g) for 72 hr [9, 13]. Animals were sacrificed following pentobarbital anesthesia (50 mg/kg ip) by exsanguination via cardiocentesis. Pancreata were immediately excised and fixed for light microscopy. Serum amylase was determined on an automated Kodak Ectachem 700 analyzer (Kodak, Rochester, NY) standardized for murine proteins. Pancreatic necrosis was established by blinded light microscopic histologic grad-
INTRODUCTION
Interleukin-1b is a primary mediator of the inflammatory response seen during acute pancreatitis [1–4]. Previous studies utilizing several distinct animal models have shown production of high levels of IL-1b within the pancreas at both the protein and mRNA levels. We have previously demonstrated pancreatic tissue levels 1
Supported by a Veteran’s Administration Merit Review Grant.
0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
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6n18$$$$$1
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01-22-97 00:25:01
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FINK ET AL.: EFFECTS OF IL-1b ON ACINAR CELLS
95
lase characteristic of severe pancreatitis (P õ 0.001 vs Time 0). IL-1ra administration decreased the maximal amylase concentration in a dose-dependent fashion with 1.0 mg/kg showing little or no effect in either model. At a dose of 10 or 100 mg/kg, however, amylase release was decreased significantly in both models (Fig. 1). Pancreatic necrosis. Histologic grading of pancreatic necrosis was defined as 0.0 in pancreatic tissue with no evidence of necrosis (Time 0) and 4.0 for the maximum necrosis present within each model (8 and 72 hr, caerulein and CDE, respectively). The degree of pancreatic necrosis correlated with the rise in serum amylase levels and again showed decreases in animals receiving IL-1ra at 10 or 100 mg/kg, but not 1.0 mg/kg (Fig. 2). In Vitro Exocrine Function and Viability
FIG. 1. Serum amylase activity during moderate and severe pancreatitis. IL-1ra at concentrations exceeding 1 mg/kg significantly attenuated serum amylase levels in both models (*P õ 0.05 vs control). ing of hematoxylin and eosin (H&E)-stained sections by a single pathologist (range 0.0 to 4.0, no evidence of necrosis and severe necrosis, respectively) as previously described [9]. Interleukin-1 blockade. One-half of the animals in each group underwent prophylactic blockade of IL-1 via ip injection of recombinant IL-1ra (Amgen, Inc., Boulder, CO) at doses of 1.0, 10, and 100 mg/kg prior to pancreatitis induction. Animals were redosed at the same concentration every 6 hr for the remainder of the experiment. In vitro exocrine function and viability: Isolation of pancreatic acini. Isolation of rat pancreatic acini was accomplished as described by Bruzzone et al. [13]. Briefly, 90 male Sprague–Dawley rats (265 { 16 g) were sacrificed via CO2 asphyxiation and the pancreata immediately excised and placed in cold Krebs–Ringer Hepes buffer (pH 7.4). Pancreata were diced into 2-mm sections and subjected to collagenase digestion utilizing type IV collagenase (Sigma, St. Louis, MO) at 1 mg/ml for 8 min. Acini were then washed twice, filtered at 200 and 70 mm, and then suspended in fresh buffer containing human albumin (0.1%). The resulting acini were plated in 24-well clusters at a density of 1 1 106 and preincubated for 30 min in the presence of 100% O2 for equilibration. Acini exocrine function and viability. Amylase release was assessed after a 30-min incubation period with increasing concentrations of recombinant IL-1b (0.1, 1.0, 10, and 100 ng/ml) (R&D Systems, Minneapolis, MN). Supernatant and whole cell fraction amylase levels were determined and expressed as percentage total amylase release. The exocrine potential of acini (positive control) was verified by amylase release at increasing doses of caerulein (10011 – 1008 M). Cell viability was determined in the presence of 100 ng/ml of IL-1b by trypan blue dye exclusion using a cell suspension containing 0.1% trypan blue dye at 0, 0.5, 1, 2, and 4 hr. All experiments were repeated a minimum of five times and the results averaged. Analysis of data. Results are expressed as means { standard error of the mean. All data analysis was performed using the EPISTAT statistical program (Epistat Services, Richardson, TX) applying the two-tailed Student t test.
RESULTS
In Vivo Pancreatitis Serum amylase. All animals receiving caerulein or the CDE diet had significant elevation of serum amy-
AID
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Exocrine function. The functional viability of acini was demonstrated by the caerulein dose–response curve which showed a typical increase in amylase release as the concentration of the CCK analogue was increased from 10011 to 1008 M. Further increases in caerulein concentrations provided the expected inhibitory effect on amylase release (Fig. 3) [14]. The addition of IL-1b at 0.1, 1.0, 10, or 100 ng/ml for 30 min had no effect on the exocrine function of isolated acini as determined by the secretion of amylase (Fig. 4). Viability. Isolated acini consistently showed greater than 93% initial viability. Cultures without additives showed a typical loss of 2–3% in viability each
FIG. 2. Histologic scoring of pancreatic necrosis. Scoring of pancreatic necrosis at Time 0 (normal pancreata) was defined as 0, while severe pancreatitis (control) for both experimental models was defined as 4.0. Blind necrosis scoring in those animals receiving IL-1ra at 10 or 100 mg/kg was significantly less than that in corresponding animals not receiving the antagonist (*P õ 0.05 vs control).
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JOURNAL OF SURGICAL RESEARCH: VOL. 67, NO. 1, JANUARY 1997
FIG. 3. Caerulein stimulation of amylase release in isolated acini. Caerulein served as the positive control demonstrating the classic rise and eventual decrease in amylase secretion as the caerulein concentration increased.
hour. The addition of IL-1b at 100 ng/ml over 4 hr had no protective or detrimental effect (Fig. 5). DISCUSSION
Interleukin-1b has a well-characterized role during inflammation including leukocyte recruitment, upregu-
FIG. 4. Exocrine function of isolated acini in the presence of IL1b. Exposure of the acini to escalating doses of recombinant IL-1b for 30 min had no detectable effect on exocrine function.
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lation of adhesion molecules, initiation of cytokine production by macrophages, and increased capillary permeability [15]. While most parenchymal cells—including pancreatic acinar cells—possess IL-1 receptors, little is known about the physiologic actions of this cytokine on cell types not commonly involved in the inflammatory response. The current study attempts to characterize the effects of IL-1b on pancreatic acini with respect to exocrine function and viability in order to define the mechanism by which IL-1 antagonism attenuates the severity of acute pancreatitis. The two models of acute pancreatitis allowed for the characterization of in vivo amylase release in the presence of IL-1 blockade. Attenuated levels of serum amylase in those animals receiving sufficient antagonist most likely reflects decreased pancreatic acinar cell damage while allowing an indirect assessment of pancreatic exocrine function. Alone, these experiments would suggest that IL-1b does have a direct effect on amylase release. The in vitro amylase secretion studies, however, demonstrate that IL-1b does not significantly effect pancreatic exocrine function and illustrate that IL-1b is not the mediator of attenuated amylase release seen during acute pancreatitis in vivo. In a similar fashion, acinar cell necrosis during acute pancreatitis was lessened with IL-1 blockade regardless of the in vivo model used. However, concentrations of IL-1b in the range of documented intrapancreatic levels seen during pancreatitis [5] had no influence on the viability of acini over the 4-hr duration of the in vitro studies. Again, these results establish that IL-1b has no direct toxic effect on pancreatic acinar cells as would be suggested by antagonism studies in live animal models.
FIG. 5. Effects of IL-1b on isolated acini viability. Initial viability of primary cultured rat acini (106/ml) was consistently ú93%. Incubation in the presence of recombinant IL-1b did not change acini viability over the 4-hr duration of the experiment.
srga
FINK ET AL.: EFFECTS OF IL-1b ON ACINAR CELLS
The significant attenuation of the severity and mortality documented in previous models of pancreatitis following IL-1 blockade has been loosely attributed to the lower number of infiltrating leukocytes and their inflammatory products as well as prevention of the direct injurious effects of IL-1b. It is now apparent that a direct detrimental effect of IL-b on acinar cells is not a major component of this mechanism. IL-1b potentiates many other physiologic responses which are essential for the propagation of pancreatitis, including the initiation of vascular permeability and ischemia–reperfusion injury within the inflamed gland [1–3]. The current study implies that inhibition of these proinflammatory properties of IL-1 corresponds to improved outcome without direct effect on acini. Clinical investigations aimed at decreasing inflammation rather than traditional approaches focusing on acinar cell function may prove beneficial. These results must also be investigated with respect to other proinflammatory cytokines since IL-1 blockade dramatically decreases the production of TNF-a and IL-6 during acute pancreatitis [8, 9] as in other acute inflammatory conditions [16, 17]. REFERENCES 1. Lowry, S. F. Cytokine mediators of immunity and inflammation. Arch. Surg. 128: 1235–1241, 1993. 2. McKay, C., Gallagher, G., Baxter, J. N., and Imrie, C. W. Systemic complications in acute pancreatitis are associated with increased monocyte cytokine release. Gut 35: A575, 1994. 3. Gross, V., Leser, H. G., Heinisch, A., and Scholmerich, J. Inflammatory mediators and cytokines—New aspects of the pathophysiology and assessment of severity of acute pancreatitis. Hepatogastroenterology 40: 522–530, 1993. 4. McKay, C., Gallagher, G., Baxter, J. N., and Imrie, C. W. Systemic complications in acute pancreatitis are associated with increased monocyte cytosine release. Gut 35: A575, 1994. 5. Norman, J., Franz, M., Riker, A., and Gower, W. R. Rapid elevation of pro-inflammatory cytokines during acute pancreatitis and their origin within the pancreas. Surg. Forum XLV: 148– 149, 1994.
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6. Norman, J., Fink, G., Franz, M., and Carter, G. Systemic cytokine gene expression induced by acute pancreatitis. Gastroenterology 108: 1236, 1995. 7. Fink, G., and Norman, J., Intrapancreatic interleukin-1b gene expression by specific leukocyte populations during acute pancreatitis. J. Surg. Res. 63: 369–373, 1996. 8. Norman, J., Fink, G., Franz, M., Guffey, J., Carter, G., Davison, B., Sexton, C., and Glaccum, M. Active interleukin-1 receptor required for maximal progression of acute pancreatitis. Ann. Surg. 223: 163–169, 1996. 9. Norman, J., Franz, M., Fink, G., Messina, J., Fabri, P. J., Gower, W. R., and Carey, L. C. Decreased mortality of severe acute pancreatitis following proximal cytokine blockade. Ann. Surg. 221: 453–460, 1995. 10. Tanaka, N., Murata, A., Uda, K., Toda, H., et al. Interleukin-1 receptor antagonist modifies the changes in vital organs induced by acute necrotizing pancreatitis in a rat experimental model. Crit. Care Med. 23: 901–908, 1995. 11. Norman, J., Fink, G., and Franz, M. Acute pancreatitis induces intrapancreatic tumor necrosis factor gene expression. Arch. Surg. 130: 966–970, 1995. 12. Tani, S., Otsuki, M., Itoh, H., Fujii, M., Nakamura, T., Oka, T., and Baba, S. Histologic and biochemical alterations in experimental acute pancreatitis induced by supramaximal caerulien stimulation. Int. J. Pancreatol. 2: 337–348, 1987. 13. Bruzzone, R., Halban, P., Gjinovci, A., and Trimble, E. A new, rapid, method for preparation of dispersed pancreatic acini. Biochem. J. 226: 621–624, 1985. 14. Leach, S. D., Modlin, I. M., Scheele, G. A., and Gorelick, F. S. Intracellular activation of digestive zymogens in rat pancreatic acini—Stimulation by high doses of cholecystokinin. J. Clin. Invest. 87: 362–366, 1991. 15. Dinarello, C. The interleukin-1 family: 10 years of discovery. FASEB J. 8: 1314–1325, 1994. 16. Granowitz, E. V., Clark, B. D., Vannier, E., Callahan, M. V., and Dinarello, C. A. Effect of interleukin-1 (IL-1) blockade on cytokine synthesis: IL-1 receptor antagonist inhibits IL-1-induced cytokine synthesis and blocks the binding of IL-1 to its type II receptor on human monocytes. Blood 79: 2356–2363, 1992. 17. Ikejima, T., Okusawa, S., Ghezzi, P., van der Meer, J. W. M., and Dinarello, C. A. Interleukin-1 induces tumor necrosis factor in human peripheral blood mononuclear cells in vitro and a circulating TNF-like activity in rabbits. J. Infect. Dis. 162: 215– 220, 1990.
srga
67, 94–97 (1997)
JR964935
Acute Pancreatitis-Induced Enzyme Release and Necrosis Are Attenuated by IL-1 Antagonism through an Indirect Mechanism GREGORY FINK, M.D., JUN YANG, M.D., GAY CARTER,
AND
JAMES NORMAN, M.D.1
Department of Surgery and the Pancreas Study Group, University of South Florida, Tampa, Florida Presented at the Annual Symposium of the Association of Veterans Administration Surgeons, Detroit, Michigan, April 28–30, 1996
in excess of 100 ng/ml during peak periods of inflammation during pancreatitis [5, 6]. Subsequent experiments have suggested that intrapancreatic IL-1b originates from within infiltrating leukocytes with no evidence available at this time to suggest production by pancreatic acinar or ductal cells [7]. Although IL-1b production correlates with pancreatic destruction, it is not felt to be just a marker of the severity of the disease, but rather an important intermediary in the progression of acute pancreatitis. Evidence for this influential role is provided by several studies demonstrating decreased mortality in lethal models of pancreatitis when IL-1b is either pharmacologically or genetically rendered inactive [8–10]. The mechanism by which IL-1b blockade improves the severity and mortality of experimental pancreatitis has not been completely delineated. We have shown that acinar cells constitutively express mRNA for both IL-1 type I and type II receptors and possess the receptors on their cell surface (unpublished data). Despite these findings, it is not known whether IL-1 serves a physiologic purpose within the pancreas at rest or during acute pancreatitis. This study was undertaken to test the hypothesis that IL-1b contributes to the severity of pancreatitis through direct effects on acinar exocrine function and detrimental effects on acinar viability.
Interleukin-1b (IL-1) is a proinflammatory cytokine which is produced within the pancreas during acute pancreatitis reaching levels which are toxic to many cell types. Since antagonism of this cytokine provides dramatic survival benefits during lethal pancreatitis, we hypothesized that IL-1 had direct secretagogue and cytolytic effects within the pancreas. The effect of IL1 on pancreatic exocrine function and tissue viability was assessed in vivo by blockade of IL-1 with varying doses of IL-1 receptor antagonist (IL-1ra) prior to the induction of either moderate (caerulein-induced) or severe (choline deficient diet-induced) necrotizing pancreatitis. Subsequent in vitro studies were conducted to determine the direct effect of IL-1 on dispersed rat acini prepared through collagenase digestion. Amylase release was measured after a 30-min incubation with varying doses of recombinant IL-1b. Viability was determined in the presence of IL-1 via trypan blue exclusion at multiple time points. Blockade of the IL-1 receptor decreased pancreatic amylase release and tissue necrosis in both models of pancreatitis in a dose-dependent fashion (1.0 mg/kg, P Å NS; 10 mg/kg, P õ 0.05; 100 mg/kg, P õ 0.05). Despite these in vivo findings, the addition of IL-1 to acini in vitro had no effect on exocrine function and failed to decrease acini viability (both, P Å NS). Pancreatic amylase release and tissue necrosis are significantly attenuated during experimental pancreatitis by IL-1 antagonism. These changes do not appear to be due to the direct action of IL-1 on pancreatic acini and are likely due to more complex interactions between acini and cytokine-producing leukocytes. q 1997 Academic Press
METHODS In vivo pancreatitis: Animal model. Animal studies were performed at an AAALAC accredited facility in accordance with the Department of Laboratory Animal Medicine at the University of South Florida. Two models of pancreatitis were utilized. A well-established model of acute moderately necrotizing pancreatitis was employed in 72 adult male NIH Swiss mice (32 { 0.3 g) by the intraperitoneal (ip) injection of caerulein (50 mg/kg/hr 1 4), a cholecystokinin analogue [11, 12]. Control mice received ip saline at the same time intervals. Animals were sacrificed at 0 and 8 hr after pancreatitis induction. In the second model, severe necrotizing pancreatitis was induced by feeding a choline-deficient ethionine-supplemented diet to young female NIH Swiss mice (15.3 { 0.2 g) for 72 hr [9, 13]. Animals were sacrificed following pentobarbital anesthesia (50 mg/kg ip) by exsanguination via cardiocentesis. Pancreata were immediately excised and fixed for light microscopy. Serum amylase was determined on an automated Kodak Ectachem 700 analyzer (Kodak, Rochester, NY) standardized for murine proteins. Pancreatic necrosis was established by blinded light microscopic histologic grad-
INTRODUCTION
Interleukin-1b is a primary mediator of the inflammatory response seen during acute pancreatitis [1–4]. Previous studies utilizing several distinct animal models have shown production of high levels of IL-1b within the pancreas at both the protein and mRNA levels. We have previously demonstrated pancreatic tissue levels 1
Supported by a Veteran’s Administration Merit Review Grant.
0022-4804/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
JSR 4935
/
6n18$$$$$1
94
01-22-97 00:25:01
srga
FINK ET AL.: EFFECTS OF IL-1b ON ACINAR CELLS
95
lase characteristic of severe pancreatitis (P õ 0.001 vs Time 0). IL-1ra administration decreased the maximal amylase concentration in a dose-dependent fashion with 1.0 mg/kg showing little or no effect in either model. At a dose of 10 or 100 mg/kg, however, amylase release was decreased significantly in both models (Fig. 1). Pancreatic necrosis. Histologic grading of pancreatic necrosis was defined as 0.0 in pancreatic tissue with no evidence of necrosis (Time 0) and 4.0 for the maximum necrosis present within each model (8 and 72 hr, caerulein and CDE, respectively). The degree of pancreatic necrosis correlated with the rise in serum amylase levels and again showed decreases in animals receiving IL-1ra at 10 or 100 mg/kg, but not 1.0 mg/kg (Fig. 2). In Vitro Exocrine Function and Viability
FIG. 1. Serum amylase activity during moderate and severe pancreatitis. IL-1ra at concentrations exceeding 1 mg/kg significantly attenuated serum amylase levels in both models (*P õ 0.05 vs control). ing of hematoxylin and eosin (H&E)-stained sections by a single pathologist (range 0.0 to 4.0, no evidence of necrosis and severe necrosis, respectively) as previously described [9]. Interleukin-1 blockade. One-half of the animals in each group underwent prophylactic blockade of IL-1 via ip injection of recombinant IL-1ra (Amgen, Inc., Boulder, CO) at doses of 1.0, 10, and 100 mg/kg prior to pancreatitis induction. Animals were redosed at the same concentration every 6 hr for the remainder of the experiment. In vitro exocrine function and viability: Isolation of pancreatic acini. Isolation of rat pancreatic acini was accomplished as described by Bruzzone et al. [13]. Briefly, 90 male Sprague–Dawley rats (265 { 16 g) were sacrificed via CO2 asphyxiation and the pancreata immediately excised and placed in cold Krebs–Ringer Hepes buffer (pH 7.4). Pancreata were diced into 2-mm sections and subjected to collagenase digestion utilizing type IV collagenase (Sigma, St. Louis, MO) at 1 mg/ml for 8 min. Acini were then washed twice, filtered at 200 and 70 mm, and then suspended in fresh buffer containing human albumin (0.1%). The resulting acini were plated in 24-well clusters at a density of 1 1 106 and preincubated for 30 min in the presence of 100% O2 for equilibration. Acini exocrine function and viability. Amylase release was assessed after a 30-min incubation period with increasing concentrations of recombinant IL-1b (0.1, 1.0, 10, and 100 ng/ml) (R&D Systems, Minneapolis, MN). Supernatant and whole cell fraction amylase levels were determined and expressed as percentage total amylase release. The exocrine potential of acini (positive control) was verified by amylase release at increasing doses of caerulein (10011 – 1008 M). Cell viability was determined in the presence of 100 ng/ml of IL-1b by trypan blue dye exclusion using a cell suspension containing 0.1% trypan blue dye at 0, 0.5, 1, 2, and 4 hr. All experiments were repeated a minimum of five times and the results averaged. Analysis of data. Results are expressed as means { standard error of the mean. All data analysis was performed using the EPISTAT statistical program (Epistat Services, Richardson, TX) applying the two-tailed Student t test.
RESULTS
In Vivo Pancreatitis Serum amylase. All animals receiving caerulein or the CDE diet had significant elevation of serum amy-
AID
JSR 4935
/
6n18$$$$$2
01-22-97 00:25:01
Exocrine function. The functional viability of acini was demonstrated by the caerulein dose–response curve which showed a typical increase in amylase release as the concentration of the CCK analogue was increased from 10011 to 1008 M. Further increases in caerulein concentrations provided the expected inhibitory effect on amylase release (Fig. 3) [14]. The addition of IL-1b at 0.1, 1.0, 10, or 100 ng/ml for 30 min had no effect on the exocrine function of isolated acini as determined by the secretion of amylase (Fig. 4). Viability. Isolated acini consistently showed greater than 93% initial viability. Cultures without additives showed a typical loss of 2–3% in viability each
FIG. 2. Histologic scoring of pancreatic necrosis. Scoring of pancreatic necrosis at Time 0 (normal pancreata) was defined as 0, while severe pancreatitis (control) for both experimental models was defined as 4.0. Blind necrosis scoring in those animals receiving IL-1ra at 10 or 100 mg/kg was significantly less than that in corresponding animals not receiving the antagonist (*P õ 0.05 vs control).
srga
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JOURNAL OF SURGICAL RESEARCH: VOL. 67, NO. 1, JANUARY 1997
FIG. 3. Caerulein stimulation of amylase release in isolated acini. Caerulein served as the positive control demonstrating the classic rise and eventual decrease in amylase secretion as the caerulein concentration increased.
hour. The addition of IL-1b at 100 ng/ml over 4 hr had no protective or detrimental effect (Fig. 5). DISCUSSION
Interleukin-1b has a well-characterized role during inflammation including leukocyte recruitment, upregu-
FIG. 4. Exocrine function of isolated acini in the presence of IL1b. Exposure of the acini to escalating doses of recombinant IL-1b for 30 min had no detectable effect on exocrine function.
AID
JSR 4935
/
6n18$$$$$2
01-22-97 00:25:01
lation of adhesion molecules, initiation of cytokine production by macrophages, and increased capillary permeability [15]. While most parenchymal cells—including pancreatic acinar cells—possess IL-1 receptors, little is known about the physiologic actions of this cytokine on cell types not commonly involved in the inflammatory response. The current study attempts to characterize the effects of IL-1b on pancreatic acini with respect to exocrine function and viability in order to define the mechanism by which IL-1 antagonism attenuates the severity of acute pancreatitis. The two models of acute pancreatitis allowed for the characterization of in vivo amylase release in the presence of IL-1 blockade. Attenuated levels of serum amylase in those animals receiving sufficient antagonist most likely reflects decreased pancreatic acinar cell damage while allowing an indirect assessment of pancreatic exocrine function. Alone, these experiments would suggest that IL-1b does have a direct effect on amylase release. The in vitro amylase secretion studies, however, demonstrate that IL-1b does not significantly effect pancreatic exocrine function and illustrate that IL-1b is not the mediator of attenuated amylase release seen during acute pancreatitis in vivo. In a similar fashion, acinar cell necrosis during acute pancreatitis was lessened with IL-1 blockade regardless of the in vivo model used. However, concentrations of IL-1b in the range of documented intrapancreatic levels seen during pancreatitis [5] had no influence on the viability of acini over the 4-hr duration of the in vitro studies. Again, these results establish that IL-1b has no direct toxic effect on pancreatic acinar cells as would be suggested by antagonism studies in live animal models.
FIG. 5. Effects of IL-1b on isolated acini viability. Initial viability of primary cultured rat acini (106/ml) was consistently ú93%. Incubation in the presence of recombinant IL-1b did not change acini viability over the 4-hr duration of the experiment.
srga
FINK ET AL.: EFFECTS OF IL-1b ON ACINAR CELLS
The significant attenuation of the severity and mortality documented in previous models of pancreatitis following IL-1 blockade has been loosely attributed to the lower number of infiltrating leukocytes and their inflammatory products as well as prevention of the direct injurious effects of IL-1b. It is now apparent that a direct detrimental effect of IL-b on acinar cells is not a major component of this mechanism. IL-1b potentiates many other physiologic responses which are essential for the propagation of pancreatitis, including the initiation of vascular permeability and ischemia–reperfusion injury within the inflamed gland [1–3]. The current study implies that inhibition of these proinflammatory properties of IL-1 corresponds to improved outcome without direct effect on acini. Clinical investigations aimed at decreasing inflammation rather than traditional approaches focusing on acinar cell function may prove beneficial. These results must also be investigated with respect to other proinflammatory cytokines since IL-1 blockade dramatically decreases the production of TNF-a and IL-6 during acute pancreatitis [8, 9] as in other acute inflammatory conditions [16, 17]. REFERENCES 1. Lowry, S. F. Cytokine mediators of immunity and inflammation. Arch. Surg. 128: 1235–1241, 1993. 2. McKay, C., Gallagher, G., Baxter, J. N., and Imrie, C. W. Systemic complications in acute pancreatitis are associated with increased monocyte cytokine release. Gut 35: A575, 1994. 3. Gross, V., Leser, H. G., Heinisch, A., and Scholmerich, J. Inflammatory mediators and cytokines—New aspects of the pathophysiology and assessment of severity of acute pancreatitis. Hepatogastroenterology 40: 522–530, 1993. 4. McKay, C., Gallagher, G., Baxter, J. N., and Imrie, C. W. Systemic complications in acute pancreatitis are associated with increased monocyte cytosine release. Gut 35: A575, 1994. 5. Norman, J., Franz, M., Riker, A., and Gower, W. R. Rapid elevation of pro-inflammatory cytokines during acute pancreatitis and their origin within the pancreas. Surg. Forum XLV: 148– 149, 1994.
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