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Leukocyte NADPH oxidase but not myeloperoxidase regulates pancreatic trypsin activation in cerulein induced pancreatitis
A170 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
953
955
ACUTE NECROTIZING PANCREATITIS IS ASSOCIATED WITH THE UPREGULATION OF SEVERAL NEUROTROPHINS. Hiroki Toma, John H. Winston, Pankaj Jay Pasricha, UTMB, Galveston, TX.
CARBONIC ANHYDRASE II AND THI TYPE CD4-POSITIVE CELLS ARE INVOLVED IN THE DEVELOPMENT OF AUTOIMMUNE·RELATED PANCREATITIS IN AN ANIMAL MODEL. Kazushige Uchida, Kazuichi Okazaki, Toshiki Nishi, Andra's Debreceni, Suguru Uose, Hiroshi Nakase, Yumi Matsushima, Chihru Kawanami, Tsutomu Chiba, Kyoto Univ, Kyoto, Japan.
The molecular mechaisms underlying the sensation of visceral pain remain obscure. Recent lines of evidence have indicated the involvement of various neruotrophins in the singnal transmission of painful information from the periphery to the brain. Methods: We used a rat model of acute necrotizing pancreatitis to investigate the changes in neurotrophins in this condition. Two intraperitoneal injections of L-arginine (L-arg) 250 mg! 100g body weight were administered to Sprague-Dawley rats (200-350 g) at an interval of one hour. Control rats received injections of vehicle. Pancreas were harvested at various timepoints from 2 hours to 10 days. Serum levels of amylase were measured and pathologic changes were scored on pancreatic sections. mRNA levels of several neurotrophins in the pancreas were examined by RNase protection assay. Results: In L-arginjected rats, significant increases in amylase levels reached a peak at one day, and thereafter declined to control values. This was accompanied by acute pathological changes consisting of severe and uniform vacuolization at 2 and 6 hours and marked inflammatory infiltrate at I day. This was followed by an almost complete necrosis of the exocrine pancreas which reached a peak at 3 and 5 days. These pathological changes were accompanied by increases in Ciliary Neurotrophic Factor (CNTF)at 2 and six hours and 3 days, Nerve Growth Factor (NGF) at 3 and 5 days, neurotrophin 3 (NT3) at 3 days, and neurotrophin 4 (NT4)at 3 and 5 days. No change in brain derived neurotrophic factor (BDNF) was observed at any timepoint. Conclusions: Several neruotrophins with known effects on sensory neurons are upregulated in pancreatitis. This may be important in the biological response to injury as well as contribute to neruonal sensitization . and persistent pain in pancreatitis.
Fold increase in mRNA levels ofneurotrophins inpancreas inL-arg-injected rats
2hours 6hours 3days 5days
CNTF
NGF
BDNF
NT3
NT4
S.9 ± 1.4" 1.8 ± 0.4" 4.4±.1.7* 1.5+0.2
0.86 ± 0.26 11.2±04 13±4.S" 4.8 + 1.3"
0.8 ± 0.23 1.1 ±0.2
0.79±0.19 1.2±0.4 4.4±1.7' 2.0±0.4
11 ± 0.4 11 ±5.S" 8.5 + 2.6"
"P
954 PANCREATIC STELLATE CELL CHARACTERIZATION IN SITU DURING EXPERIMENTAL PANCREATITIS. S. Turi, W. Domschke, M. M. Lerch, Dept of Medicine B, Muenster Univ, Muenster, Germany. The development of chronic pancreatitis involves the deposition of extracellular matrix in the pancreas. Interstitial stellate cells (Ito cells) are thought to represent a major source of this extracellular matrix production. We have studied the proliferation and activation of cells that carry stellate cell markers in two animal models of pancreatitis. Methods: Edematous pancreatitis was induced in rats by i.p. caerulein (40 micro g/kg at Oh and 3h). Hemorrhagic-necrotizing pancreatitis was induced by coadministration of the bradykinin antagonist HOE 140 (O,lmg/kg at 30min and 3h). Animals were sacrificed at intervals up to 90 days. Cryosections were prepared from pancreatic tissue and immunolabelled with antibodies against desmin, glial fibrillary acidic protein (GFAP) and a-smooth muscle actin (a-SMA). Micrographs were evaluated by morphometry in a blinded fashion. A minimum of 4000 cells were studied for each experiment and data were expressed in percent of all cells z SEM. Biochemical parameters were assayed using standard protocols. Results: Serum amylase levels increased irrespective of the variety of pancreatitis. Acinar cell necrosis in the caerulein group was 7% and in the necrotizing pancreatitis group exceeded 30%. 6.4% ::': 0.4 of cells from control animals were desmin positive. Their number increased significantly to a maximum of 10.8% ::': 0.4 in the necrotizing pancreatitis group at day 2 but to only 7.8% ::': 0.6 (n.s.) in the caerulein group. 1.9% ::': 0.2 of cells were GFAP positive in controls. Their number increased to 4.9 % ::': 0.5 in the necrotizing pancreatitis group and to 3.9% ::': 0.6 in the caerulein group (both p
Background and Aim: It has been reported that an autoimmune mechanism may be involved in some patients with idiopathic chronic pancreatitis, which is so-called "autoimmune pancreatitis (AlP)". We have reported that autoantibodies against carbonic anhydrase II (CA-II) were frequently identified in these patients. However, it is still unknown whether CA-II plays an important role in the pathogenesis or not. To clarify it, we established an animal model of AlP using neonatally thymectomized (nTx) mice. Materials and Methods: BALBfc mice (n=IO) were thymectomized on the 3rd day after birth. At the period of 8 weeks old, mice were subcutaneously immunized with the emulsion of CA-II (O.lmg) and Freund's complete adjuvant, and sacrificed after 3 times booster injections. Normal mice immunized with or without CA-II and nTx-mice injected with adjuvant alone were served as controls (n=5 in each group). Moreover, we transferred the whole, CD4- or CD8-positive spleen cells (lxl06 ) prepared from CA-II immunized or non-immunized nTx-mice to nude mice (n =5 in each group). After 3 months, we sacrificed the transferred mice. We studied histological findings of the pancreas and salivary gland. The subsets of infiltrating lymphocytes were analyzed by immunohistochemistry and flow-cytometry. Gene expressions of IFN-y.md IL-4 were analyzed by RT-PCR. Results: Histological findings showed infiltration of lymphocytes around the duct of the pancreas (in 6 of 10) and salivary gland (4 of 10) in immunized nTx-mice. All of the nude mice transferred whole spleen cells or CD4-positive cells prepared from immunized nTx-mice showed infiltration of lymphocytes around the duct in the pancreas and salivary gland. On the other hand, none of the mice transferred with CD8-positive cells or non-immunized spleen cells showed infiltration of the lymphocytes. Immunohistochemistry and flow-cytometric analysis showed most of infiltrating lymphocytes were CD4-positive cells. Expression of IFN--ygene was up regulated in the nude mice transferred whole or CD4-positive cells prepared from immunized nTx-mice. Conclusion: An autoimmune mechanism against CA-II is involved in the pathogenesis of this model, in which effecter cells are Thl-type CD4-positive cells.
956 LEUKOCYTE NADPH OXIDASE BUT NOT MYELOPEROXI· DASE REGULATES PANCREATIC TRYPSIN ACTIVATION IN CERULEIN INDUCED PANCREATITIS. E. Vaquero, A. S. Gukovskaya, M. L. Brennan, A. 1. Lusis, S. M. Holland, S. 1. Pandol, UCLA & VA Greater Los Angeles Healthcare System, Los Angeles, CA; Univ of CA, Los Angeles, Los Angeles, CA; NIAID, NIH, Bethesda, MD. Recently, we showed that neutrophils regulate the severity of rat cerulein pancreatitis. We found that neutrophil depletion resulted in improvement in parameters of pancreatitis such as hyperamylasemia and hyperlipasemia. We also found that one of the key early events in cerulein-induced pancreatitis, pancreatic trypsin activation, was much less in neutrophildepleted than in non-depleted rats. The present study aimed to determine the mechanisms of neutrophil effects on trypsin activation in pancreatitis. For this purpose, we studied cerulein-induced pancreatitis in two types of knockout mice: one, p47 (phox-f-), deficient in nicotinamide dinucleotide phosphate (NADPH) oxydase, and another, MPO -!-, deficient in myeloperoxidase (MPO). Leukocyte NADPH oxidase is a major producer of superoxide, hydrogen peroxide, and hydroxyl radical, key reactive oxygen species (ROS). MPO further converts H 2 0 2 into hypochlorous acid. Pancreatitis was induced in 4 groups of mice: NADPH oxidase wild-type (NDfwt), NADPH oxidase knockout (ND/ko), MPO wild-type (MPOfwt), and MPO knockout (MPO/ko), by 7 hourly i.p, injections of cerulein. Saline injected mice served as controls. Animals were sacrificed 1 h after the last injection. Trypsin activity and total trypsinogen were measured in the pancreas by a fluorogenic assay, and serum amylase and lipase by enzymatic assay. In NDfwt mice, cerulein induced a 7.5-fold activation of pancreatic trypsin compared to control. In contrast, in ND/ko mice cerulein did not significantly increase trypsin activity. Similarly, intrapancreatic trypsin/trypsinogen ratio significantly increased in the NDfwt cerulein group compared to NDfwt saline group, ND/ko cerulein group, and ND/ko saline group (p < 0.02). In contrast, cerulein-induced trypsin activation was not different between the MPOfwt and MPO/ko groups. This may indicate that hypochlorous acid does not participate in regulation of trypsin activity by neutrophils. Interestingly, the increase in serum amylase and lipase was the same in cerulein-treated animals from both wild type and knockout ND or MPO mice. The results provide new evidence on leukocyte regulation of pancreatic trypsin activity in cerulein pancreatitis. Furthermore, they demonstrate that NADPH oxidase mediates the effect of leukocytes on trypsin activation. This suggests the involvement of ROS in cerulein induced trypsin activation.
GASTROENTEROLOGY Vol. 118, No.4
953
955
ACUTE NECROTIZING PANCREATITIS IS ASSOCIATED WITH THE UPREGULATION OF SEVERAL NEUROTROPHINS. Hiroki Toma, John H. Winston, Pankaj Jay Pasricha, UTMB, Galveston, TX.
CARBONIC ANHYDRASE II AND THI TYPE CD4-POSITIVE CELLS ARE INVOLVED IN THE DEVELOPMENT OF AUTOIMMUNE·RELATED PANCREATITIS IN AN ANIMAL MODEL. Kazushige Uchida, Kazuichi Okazaki, Toshiki Nishi, Andra's Debreceni, Suguru Uose, Hiroshi Nakase, Yumi Matsushima, Chihru Kawanami, Tsutomu Chiba, Kyoto Univ, Kyoto, Japan.
The molecular mechaisms underlying the sensation of visceral pain remain obscure. Recent lines of evidence have indicated the involvement of various neruotrophins in the singnal transmission of painful information from the periphery to the brain. Methods: We used a rat model of acute necrotizing pancreatitis to investigate the changes in neurotrophins in this condition. Two intraperitoneal injections of L-arginine (L-arg) 250 mg! 100g body weight were administered to Sprague-Dawley rats (200-350 g) at an interval of one hour. Control rats received injections of vehicle. Pancreas were harvested at various timepoints from 2 hours to 10 days. Serum levels of amylase were measured and pathologic changes were scored on pancreatic sections. mRNA levels of several neurotrophins in the pancreas were examined by RNase protection assay. Results: In L-arginjected rats, significant increases in amylase levels reached a peak at one day, and thereafter declined to control values. This was accompanied by acute pathological changes consisting of severe and uniform vacuolization at 2 and 6 hours and marked inflammatory infiltrate at I day. This was followed by an almost complete necrosis of the exocrine pancreas which reached a peak at 3 and 5 days. These pathological changes were accompanied by increases in Ciliary Neurotrophic Factor (CNTF)at 2 and six hours and 3 days, Nerve Growth Factor (NGF) at 3 and 5 days, neurotrophin 3 (NT3) at 3 days, and neurotrophin 4 (NT4)at 3 and 5 days. No change in brain derived neurotrophic factor (BDNF) was observed at any timepoint. Conclusions: Several neruotrophins with known effects on sensory neurons are upregulated in pancreatitis. This may be important in the biological response to injury as well as contribute to neruonal sensitization . and persistent pain in pancreatitis.
Fold increase in mRNA levels ofneurotrophins inpancreas inL-arg-injected rats
2hours 6hours 3days 5days
CNTF
NGF
BDNF
NT3
NT4
S.9 ± 1.4" 1.8 ± 0.4" 4.4±.1.7* 1.5+0.2
0.86 ± 0.26 11.2±04 13±4.S" 4.8 + 1.3"
0.8 ± 0.23 1.1 ±0.2
0.79±0.19 1.2±0.4 4.4±1.7' 2.0±0.4
11 ± 0.4 11 ±5.S" 8.5 + 2.6"
"P
954 PANCREATIC STELLATE CELL CHARACTERIZATION IN SITU DURING EXPERIMENTAL PANCREATITIS. S. Turi, W. Domschke, M. M. Lerch, Dept of Medicine B, Muenster Univ, Muenster, Germany. The development of chronic pancreatitis involves the deposition of extracellular matrix in the pancreas. Interstitial stellate cells (Ito cells) are thought to represent a major source of this extracellular matrix production. We have studied the proliferation and activation of cells that carry stellate cell markers in two animal models of pancreatitis. Methods: Edematous pancreatitis was induced in rats by i.p. caerulein (40 micro g/kg at Oh and 3h). Hemorrhagic-necrotizing pancreatitis was induced by coadministration of the bradykinin antagonist HOE 140 (O,lmg/kg at 30min and 3h). Animals were sacrificed at intervals up to 90 days. Cryosections were prepared from pancreatic tissue and immunolabelled with antibodies against desmin, glial fibrillary acidic protein (GFAP) and a-smooth muscle actin (a-SMA). Micrographs were evaluated by morphometry in a blinded fashion. A minimum of 4000 cells were studied for each experiment and data were expressed in percent of all cells z SEM. Biochemical parameters were assayed using standard protocols. Results: Serum amylase levels increased irrespective of the variety of pancreatitis. Acinar cell necrosis in the caerulein group was 7% and in the necrotizing pancreatitis group exceeded 30%. 6.4% ::': 0.4 of cells from control animals were desmin positive. Their number increased significantly to a maximum of 10.8% ::': 0.4 in the necrotizing pancreatitis group at day 2 but to only 7.8% ::': 0.6 (n.s.) in the caerulein group. 1.9% ::': 0.2 of cells were GFAP positive in controls. Their number increased to 4.9 % ::': 0.5 in the necrotizing pancreatitis group and to 3.9% ::': 0.6 in the caerulein group (both p
Background and Aim: It has been reported that an autoimmune mechanism may be involved in some patients with idiopathic chronic pancreatitis, which is so-called "autoimmune pancreatitis (AlP)". We have reported that autoantibodies against carbonic anhydrase II (CA-II) were frequently identified in these patients. However, it is still unknown whether CA-II plays an important role in the pathogenesis or not. To clarify it, we established an animal model of AlP using neonatally thymectomized (nTx) mice. Materials and Methods: BALBfc mice (n=IO) were thymectomized on the 3rd day after birth. At the period of 8 weeks old, mice were subcutaneously immunized with the emulsion of CA-II (O.lmg) and Freund's complete adjuvant, and sacrificed after 3 times booster injections. Normal mice immunized with or without CA-II and nTx-mice injected with adjuvant alone were served as controls (n=5 in each group). Moreover, we transferred the whole, CD4- or CD8-positive spleen cells (lxl06 ) prepared from CA-II immunized or non-immunized nTx-mice to nude mice (n =5 in each group). After 3 months, we sacrificed the transferred mice. We studied histological findings of the pancreas and salivary gland. The subsets of infiltrating lymphocytes were analyzed by immunohistochemistry and flow-cytometry. Gene expressions of IFN-y.md IL-4 were analyzed by RT-PCR. Results: Histological findings showed infiltration of lymphocytes around the duct of the pancreas (in 6 of 10) and salivary gland (4 of 10) in immunized nTx-mice. All of the nude mice transferred whole spleen cells or CD4-positive cells prepared from immunized nTx-mice showed infiltration of lymphocytes around the duct in the pancreas and salivary gland. On the other hand, none of the mice transferred with CD8-positive cells or non-immunized spleen cells showed infiltration of the lymphocytes. Immunohistochemistry and flow-cytometric analysis showed most of infiltrating lymphocytes were CD4-positive cells. Expression of IFN--ygene was up regulated in the nude mice transferred whole or CD4-positive cells prepared from immunized nTx-mice. Conclusion: An autoimmune mechanism against CA-II is involved in the pathogenesis of this model, in which effecter cells are Thl-type CD4-positive cells.
956 LEUKOCYTE NADPH OXIDASE BUT NOT MYELOPEROXI· DASE REGULATES PANCREATIC TRYPSIN ACTIVATION IN CERULEIN INDUCED PANCREATITIS. E. Vaquero, A. S. Gukovskaya, M. L. Brennan, A. 1. Lusis, S. M. Holland, S. 1. Pandol, UCLA & VA Greater Los Angeles Healthcare System, Los Angeles, CA; Univ of CA, Los Angeles, Los Angeles, CA; NIAID, NIH, Bethesda, MD. Recently, we showed that neutrophils regulate the severity of rat cerulein pancreatitis. We found that neutrophil depletion resulted in improvement in parameters of pancreatitis such as hyperamylasemia and hyperlipasemia. We also found that one of the key early events in cerulein-induced pancreatitis, pancreatic trypsin activation, was much less in neutrophildepleted than in non-depleted rats. The present study aimed to determine the mechanisms of neutrophil effects on trypsin activation in pancreatitis. For this purpose, we studied cerulein-induced pancreatitis in two types of knockout mice: one, p47 (phox-f-), deficient in nicotinamide dinucleotide phosphate (NADPH) oxydase, and another, MPO -!-, deficient in myeloperoxidase (MPO). Leukocyte NADPH oxidase is a major producer of superoxide, hydrogen peroxide, and hydroxyl radical, key reactive oxygen species (ROS). MPO further converts H 2 0 2 into hypochlorous acid. Pancreatitis was induced in 4 groups of mice: NADPH oxidase wild-type (NDfwt), NADPH oxidase knockout (ND/ko), MPO wild-type (MPOfwt), and MPO knockout (MPO/ko), by 7 hourly i.p, injections of cerulein. Saline injected mice served as controls. Animals were sacrificed 1 h after the last injection. Trypsin activity and total trypsinogen were measured in the pancreas by a fluorogenic assay, and serum amylase and lipase by enzymatic assay. In NDfwt mice, cerulein induced a 7.5-fold activation of pancreatic trypsin compared to control. In contrast, in ND/ko mice cerulein did not significantly increase trypsin activity. Similarly, intrapancreatic trypsin/trypsinogen ratio significantly increased in the NDfwt cerulein group compared to NDfwt saline group, ND/ko cerulein group, and ND/ko saline group (p < 0.02). In contrast, cerulein-induced trypsin activation was not different between the MPOfwt and MPO/ko groups. This may indicate that hypochlorous acid does not participate in regulation of trypsin activity by neutrophils. Interestingly, the increase in serum amylase and lipase was the same in cerulein-treated animals from both wild type and knockout ND or MPO mice. The results provide new evidence on leukocyte regulation of pancreatic trypsin activity in cerulein pancreatitis. Furthermore, they demonstrate that NADPH oxidase mediates the effect of leukocytes on trypsin activation. This suggests the involvement of ROS in cerulein induced trypsin activation.