- Email: [email protected]
Exocrine pancreatitis and sialoadenitis in mice induced by a murine retrovirus: Analysis of cytokine production in organ lesions in situ
April 2000
AGAAl71
957
959
MAP KINASE PHOSPHATASES (MKPS) ARE EARLY RESPON· SIVE GENES DURING INDUCTION OF CERULEIN HYPER· STIMULATION PANCREATITIS. Thomas Hofken, Nadine Keller, Goke Burkhard, Andreas C. Wagner, Dept of Gastroenterology, Inselspital, Bern, Switzerland. The recently discovered Map kinase phosphatases (MKPs) downregulate MAP-kinases such as ERK, p38 and JNK. This may be important to better understand the pancreatic stress reaction since pancreatic MAP-kinases are rapidly induced following secretagogue stimulation, JNK is activated only following induction of pancreatitis and, JNK inhibition ameliorates pancreatitis. We, therefore, investigated expression and regulation of pancreatic MKPs. Methods: Following iv application of cerulein, rat pancreatic MKP expression in vivo was studied using northern analysis. Effects of MKPI inhibition on JNK activation were tested in vitro in acutely isolated acini. Results: While expressed at or below the detection threshold in control animals, pancreatic mRNA levels of MKPI, MKP3 and MKP5 were strongly induced following cerulein hyperstimulation but not without induction of pancreatitis. MKP5 expression occured most rapidly, peaking 30 min after cerulein treatment, while MKPI and MKP3 peaked at 60 min. MKP mRNA levels returned to near basal levels within 3-4 hours. In contrast, MKP4 was not expressed in the pancreas while MKPx and VHR likely are constitutively expressed but not regulated through cerulein. Interestingly, inhibition of MKPI by Ro-31-8220 maximally induced JNK but not p38 or ERK activation in acutely isolated acini while a combination of cerulein with Ro-31-8220 was not additive. Conclusion: This is the first demonstration of MKP expression and regulation in an animal model. MKP1, MKP3 and MKP5 clearly are early responsive genes during pancreatitis induction. Evidence from cell culture systems indicates that MKP's act as negative regulators of MAP-kinases. Given the time course and dose response of cerulein-induced MKP mRNA expression and Map kinase activation, this paradigm may hold true for pancreatic stress episodes such as hyperstimulation in vivo. Although the exact roles of individual MKPs are unclear, we propose a hypothetical model in which pancreatic stress activates stress kinases such as JNK or other kinases such as ERK or p38. As a negative feedback, MKP mRNA expression is then induced, possibly by activation of transcription factors through Map kinases. MKP then downregulate Map kinases. Effects of MKPI inhibition through Ro-31-8220 further lead us to hypothesize that JNK possibly is a preferred substrate of MKPI and that low levels of pancreatic MKPI expression may tonically suppress JNK activation, which is, therefore, not measurable in unstimulated animals.
IDENTIFICATION OF THE CA2+·REGULATED CYTOSOLIC CYSTEIN PROTEASE CALPAIN IN RAT PANCREATIC ACINAR CELLS. Heike Weber, Hans-Heinrich Hopp, Thomas Noack, Peter Schuff-Werner, Inst of Clin Chemistry and Pathobiochemistry, Univ of Rostock, Rostock, Germany; Institute of Physiology, Univ of Rostock, Rostock, Germany. Background/Aims: The nonlysosomal Ca2+-activated cystein proteases calpains (EC 3.4.22.17) have been suggested to playa regulatory role in the metabolism of membrane and cytoskeletal proteins. However, their exact functions have not yet been established. Whereas two kinds of ubiquitous calpains with different Ca2+ sensitivities are found in various mammalian cells, they are not detected in pancreatic acinar cells. Therefore, our aims were to prove whether calpains are localized in isolated rat pancreatic acinar cells and whether they play a role in the acinar secretory process. Methods: Calpain was detected immunocytochemically. In addition, the Ca2+-mediated calpain activation in the presence and absence of the Ca2+ chelator BAPTA-AM (20 pM), of extracellular Ca2+ and of the calpain inhibitor N-CBZ-(Leu)z-Tyr-CHN z (CBZ; 100 p.M), respectively, was measured spectrophotometrically and by single cell fluorimetry, using the fluorescent calpain substrate Suc-(Leu)z-Val-Tyr-AMC (50 p.M). Ionomycin, acetylcholine (0.1 p.M) and ATP (10 p.M) were used as Caz+ modulators. The role on enzyme secretion was studied by examining the effect of CBZ on the amylase release of isolated pancreatic acini stimulated with 100 pM CCK. Results: Immunofluorescence staining of acinar cells showed a diffuse cytosolic localization of calpain. Treatment of acinar cells (l06/ml) with ionomycin (p.M: 1.25; 2.5) induced a significant dosedependent cleavage of the calpain substrate in comparison with the control within 2 min. This enzyme activation was completely prevented by BAPTA-AM, Ca2+-free medium and CBZ. The experiments on single acini using ionomycin (200 p.M), ATP and acetylcholine, respectively, revealed comparable results and showed that the enzyme was activated immediately after increasing the cytosolic Ca2+ concentration. Furthermore, we found that the CCK-stimulated amylase secretion was significantly reduced by the calpain inhibitor. Conclusions: Our results show that the cytosolic cystein protease calpain exists in pancreatic acinar cells. The regulation of the calpain activity by Caz+ and the regulatory effect on pancreatic enzyme secretion indicate that the protease may play a role in signal transduction. The findings also show that a calpain activation induced by an unphysiological increase in the cytosolic Caz+ caused alterations of acinar cells. We therefore assume that this mechanism may play a role in the development of acute pancreatitis.
958
967
EXOCRINE PANCREATITIS AND SIALOADENITIS IN MICE IN· DUCED BY A MURINE RETROVIRUS: ANALYSIS OF CYTO· KINE PRODUCTION IN ORGAN LESIONS IN SITU. S. Watanabe, K. Suzuki, H. Suriki, Y. Baba, S. Sasaki, H. Kawacbi, F. Shimizu, H. Asakura, Niigata Univ, Niigata, Japan. BACKGROUND: We previously reported that Sjoegren's syndrome(SjS) like sialoadenitis and exocrine pancreatitis were induced in mice infected with LP-BM5 murine leukemia virus (Lab Invest 1993). The virus is known to induce severe immunodeficiency termed as murine AIDS (MAIDS).We also revealed that adoptive transfer of splenocytes of MAIDS mice could induce inflammatory bowel disease-like colitis as well as SjS-like exocrinopathy in nude mice (Gut 1997). Cytokine imbalance between Thl and Th2 CD4+ T cells is now considered to play an important role in the development of some autoimmune diseases. AIM: To determine Th-I v.s. Th-2 skewing of cytokine production in exocrinopathy in mice with MAIDS. METHODS: Four-week-old B6 mice were inoculated intraperitoneally with LP-BM5, and the mice were killed to examine the exocrinopathy lesions. Untreated B6 mice were served as control. Histopathological, immunohistochemical and flow cytometric analyses were performed on the pancreas and salivary glands of mice. In addition, expression of mRNA of lPN-gamma and IL-1O was detected by RT-PCR and protein expression of these cytokines was also studied by double-color staining immunofluorescence technique. RESULTS: All of the mice with MAIDS developed SjS-like exocrine pancreatitis and sialoadenitis. IF study and flow cytometric analyses showed that not CD8+ T cells but CD4+ cells, Mac- 1+ cells and B220+ cells, were detected in exocrine glands lesions. mRNA and proteins of lPN-gamma and IL- 10 were mainly detected on CD4+ T cells and Mac-I + cells infiltrating in the organ lesions. As for CD4 + T cells, any skewing of Th 1 or Th2 was not detected. These pathological organ lesions were not observed in control mice. CONCLUSION: These results indicate that CD4 + T cells of MAIDS mice playa crucial role in induction of exocrinopathy by producing lPN-gamma and IL- 10, and that Mac- I + cells of MAIDS mice exaggerate the lesions.
HEUCOBACTER PYLORI NEGATIVELY SELECTS T CELL RE· SPONSES THROUGH FASIFAS LIGAND INTERACTIONS. Jide Wang, Edwards G. Brooks, Timothy L. Denning, Jacques Pappo, Peter B. Ernst, UTMB, Galveston, TX; AstraZeneca Research Ctr, Boston, MA. Background: Humans infected with Helicobacter pylori (H. pylori) cannot clear the infection implying that the bacteria have evaded the host response. Studies have shown that H. pylori inhibits the growth of T cells in vitro leading to the suggestion that infection imparts an immunological tolerance that may impair the development of protective immunity. To investigate the mechanism by which H. pylori inhibits T cell growth and prevents immunity, the effect of the bacteria on FaslFas ligand expression and T cell apoptosis was examined. Methods: T cell lines derived from peripheral blood or the gastric mucosa were exposed to H. pylori and apoptosis was evaluated by flow cytometry or by detecting DNA fragmentation. FaslFasL interactions were implicated by detecting the expression of Fas and FasL mRNA and protein by resting or H. pylori-stimulated T cells as well as by specifically inhibiting Fas-mediated killing. Results: Apoptotic T cell death in 50% of the T cells was caused by H. pylori but not Campylobacter jejuni as evidenced by DNA fragmentation and increased Annexin V binding. H. pylori strains bearing the cag pathogenicity island induced T cell death while isogenic mutants lacking these genes did not. H. pylori induced apoptosis in three T cell lines and this response was blocked by anti-Fas antibody (ZB4). Engagement of Fas leads to the activation of caspase 8 via FADD (Pas-associated death domain) and the eventual triggering of other downstream caspases that lead to DNA degradation. Thus, we showed that a caspase 8 inhibitor (100 J-tM Z-IETD) inhibited the H. pylori-induced apoptosis. Furthermore, a T cell line in which Fas was rendered nonfunctional by a frame shift mutation affecting the cytoplasmic domain was resistant to H. pylori-induced death. The expression of mRNA and protein for FasL on T cell lines was up-regulated by H. pylori and temporally related to H. pylori-mediated killing. Moreover, inhibiting protein synthesis with 10 mM emitine entirely blocked FasL expression and the induction of apoptosis in the T cells after stimulation with H. pylori. Preventing the cleavage of FasL with a metalloproteinase inhibitortl.Itl-phenanthroline)increased H. pylori-mediated killing. Conclusion: H. pylori induces the suicide or fratricide of Pas-bearing T cells through the induction of FasL expression. Thus, persistent infection with strains bearing the cag pathogenicity island may be favored by the induction of local tolerance through the negative selection of T cells encountering H. pylori antigens.
AGAAl71
957
959
MAP KINASE PHOSPHATASES (MKPS) ARE EARLY RESPON· SIVE GENES DURING INDUCTION OF CERULEIN HYPER· STIMULATION PANCREATITIS. Thomas Hofken, Nadine Keller, Goke Burkhard, Andreas C. Wagner, Dept of Gastroenterology, Inselspital, Bern, Switzerland. The recently discovered Map kinase phosphatases (MKPs) downregulate MAP-kinases such as ERK, p38 and JNK. This may be important to better understand the pancreatic stress reaction since pancreatic MAP-kinases are rapidly induced following secretagogue stimulation, JNK is activated only following induction of pancreatitis and, JNK inhibition ameliorates pancreatitis. We, therefore, investigated expression and regulation of pancreatic MKPs. Methods: Following iv application of cerulein, rat pancreatic MKP expression in vivo was studied using northern analysis. Effects of MKPI inhibition on JNK activation were tested in vitro in acutely isolated acini. Results: While expressed at or below the detection threshold in control animals, pancreatic mRNA levels of MKPI, MKP3 and MKP5 were strongly induced following cerulein hyperstimulation but not without induction of pancreatitis. MKP5 expression occured most rapidly, peaking 30 min after cerulein treatment, while MKPI and MKP3 peaked at 60 min. MKP mRNA levels returned to near basal levels within 3-4 hours. In contrast, MKP4 was not expressed in the pancreas while MKPx and VHR likely are constitutively expressed but not regulated through cerulein. Interestingly, inhibition of MKPI by Ro-31-8220 maximally induced JNK but not p38 or ERK activation in acutely isolated acini while a combination of cerulein with Ro-31-8220 was not additive. Conclusion: This is the first demonstration of MKP expression and regulation in an animal model. MKP1, MKP3 and MKP5 clearly are early responsive genes during pancreatitis induction. Evidence from cell culture systems indicates that MKP's act as negative regulators of MAP-kinases. Given the time course and dose response of cerulein-induced MKP mRNA expression and Map kinase activation, this paradigm may hold true for pancreatic stress episodes such as hyperstimulation in vivo. Although the exact roles of individual MKPs are unclear, we propose a hypothetical model in which pancreatic stress activates stress kinases such as JNK or other kinases such as ERK or p38. As a negative feedback, MKP mRNA expression is then induced, possibly by activation of transcription factors through Map kinases. MKP then downregulate Map kinases. Effects of MKPI inhibition through Ro-31-8220 further lead us to hypothesize that JNK possibly is a preferred substrate of MKPI and that low levels of pancreatic MKPI expression may tonically suppress JNK activation, which is, therefore, not measurable in unstimulated animals.
IDENTIFICATION OF THE CA2+·REGULATED CYTOSOLIC CYSTEIN PROTEASE CALPAIN IN RAT PANCREATIC ACINAR CELLS. Heike Weber, Hans-Heinrich Hopp, Thomas Noack, Peter Schuff-Werner, Inst of Clin Chemistry and Pathobiochemistry, Univ of Rostock, Rostock, Germany; Institute of Physiology, Univ of Rostock, Rostock, Germany. Background/Aims: The nonlysosomal Ca2+-activated cystein proteases calpains (EC 3.4.22.17) have been suggested to playa regulatory role in the metabolism of membrane and cytoskeletal proteins. However, their exact functions have not yet been established. Whereas two kinds of ubiquitous calpains with different Ca2+ sensitivities are found in various mammalian cells, they are not detected in pancreatic acinar cells. Therefore, our aims were to prove whether calpains are localized in isolated rat pancreatic acinar cells and whether they play a role in the acinar secretory process. Methods: Calpain was detected immunocytochemically. In addition, the Ca2+-mediated calpain activation in the presence and absence of the Ca2+ chelator BAPTA-AM (20 pM), of extracellular Ca2+ and of the calpain inhibitor N-CBZ-(Leu)z-Tyr-CHN z (CBZ; 100 p.M), respectively, was measured spectrophotometrically and by single cell fluorimetry, using the fluorescent calpain substrate Suc-(Leu)z-Val-Tyr-AMC (50 p.M). Ionomycin, acetylcholine (0.1 p.M) and ATP (10 p.M) were used as Caz+ modulators. The role on enzyme secretion was studied by examining the effect of CBZ on the amylase release of isolated pancreatic acini stimulated with 100 pM CCK. Results: Immunofluorescence staining of acinar cells showed a diffuse cytosolic localization of calpain. Treatment of acinar cells (l06/ml) with ionomycin (p.M: 1.25; 2.5) induced a significant dosedependent cleavage of the calpain substrate in comparison with the control within 2 min. This enzyme activation was completely prevented by BAPTA-AM, Ca2+-free medium and CBZ. The experiments on single acini using ionomycin (200 p.M), ATP and acetylcholine, respectively, revealed comparable results and showed that the enzyme was activated immediately after increasing the cytosolic Ca2+ concentration. Furthermore, we found that the CCK-stimulated amylase secretion was significantly reduced by the calpain inhibitor. Conclusions: Our results show that the cytosolic cystein protease calpain exists in pancreatic acinar cells. The regulation of the calpain activity by Caz+ and the regulatory effect on pancreatic enzyme secretion indicate that the protease may play a role in signal transduction. The findings also show that a calpain activation induced by an unphysiological increase in the cytosolic Caz+ caused alterations of acinar cells. We therefore assume that this mechanism may play a role in the development of acute pancreatitis.
958
967
EXOCRINE PANCREATITIS AND SIALOADENITIS IN MICE IN· DUCED BY A MURINE RETROVIRUS: ANALYSIS OF CYTO· KINE PRODUCTION IN ORGAN LESIONS IN SITU. S. Watanabe, K. Suzuki, H. Suriki, Y. Baba, S. Sasaki, H. Kawacbi, F. Shimizu, H. Asakura, Niigata Univ, Niigata, Japan. BACKGROUND: We previously reported that Sjoegren's syndrome(SjS) like sialoadenitis and exocrine pancreatitis were induced in mice infected with LP-BM5 murine leukemia virus (Lab Invest 1993). The virus is known to induce severe immunodeficiency termed as murine AIDS (MAIDS).We also revealed that adoptive transfer of splenocytes of MAIDS mice could induce inflammatory bowel disease-like colitis as well as SjS-like exocrinopathy in nude mice (Gut 1997). Cytokine imbalance between Thl and Th2 CD4+ T cells is now considered to play an important role in the development of some autoimmune diseases. AIM: To determine Th-I v.s. Th-2 skewing of cytokine production in exocrinopathy in mice with MAIDS. METHODS: Four-week-old B6 mice were inoculated intraperitoneally with LP-BM5, and the mice were killed to examine the exocrinopathy lesions. Untreated B6 mice were served as control. Histopathological, immunohistochemical and flow cytometric analyses were performed on the pancreas and salivary glands of mice. In addition, expression of mRNA of lPN-gamma and IL-1O was detected by RT-PCR and protein expression of these cytokines was also studied by double-color staining immunofluorescence technique. RESULTS: All of the mice with MAIDS developed SjS-like exocrine pancreatitis and sialoadenitis. IF study and flow cytometric analyses showed that not CD8+ T cells but CD4+ cells, Mac- 1+ cells and B220+ cells, were detected in exocrine glands lesions. mRNA and proteins of lPN-gamma and IL- 10 were mainly detected on CD4+ T cells and Mac-I + cells infiltrating in the organ lesions. As for CD4 + T cells, any skewing of Th 1 or Th2 was not detected. These pathological organ lesions were not observed in control mice. CONCLUSION: These results indicate that CD4 + T cells of MAIDS mice playa crucial role in induction of exocrinopathy by producing lPN-gamma and IL- 10, and that Mac- I + cells of MAIDS mice exaggerate the lesions.
HEUCOBACTER PYLORI NEGATIVELY SELECTS T CELL RE· SPONSES THROUGH FASIFAS LIGAND INTERACTIONS. Jide Wang, Edwards G. Brooks, Timothy L. Denning, Jacques Pappo, Peter B. Ernst, UTMB, Galveston, TX; AstraZeneca Research Ctr, Boston, MA. Background: Humans infected with Helicobacter pylori (H. pylori) cannot clear the infection implying that the bacteria have evaded the host response. Studies have shown that H. pylori inhibits the growth of T cells in vitro leading to the suggestion that infection imparts an immunological tolerance that may impair the development of protective immunity. To investigate the mechanism by which H. pylori inhibits T cell growth and prevents immunity, the effect of the bacteria on FaslFas ligand expression and T cell apoptosis was examined. Methods: T cell lines derived from peripheral blood or the gastric mucosa were exposed to H. pylori and apoptosis was evaluated by flow cytometry or by detecting DNA fragmentation. FaslFasL interactions were implicated by detecting the expression of Fas and FasL mRNA and protein by resting or H. pylori-stimulated T cells as well as by specifically inhibiting Fas-mediated killing. Results: Apoptotic T cell death in 50% of the T cells was caused by H. pylori but not Campylobacter jejuni as evidenced by DNA fragmentation and increased Annexin V binding. H. pylori strains bearing the cag pathogenicity island induced T cell death while isogenic mutants lacking these genes did not. H. pylori induced apoptosis in three T cell lines and this response was blocked by anti-Fas antibody (ZB4). Engagement of Fas leads to the activation of caspase 8 via FADD (Pas-associated death domain) and the eventual triggering of other downstream caspases that lead to DNA degradation. Thus, we showed that a caspase 8 inhibitor (100 J-tM Z-IETD) inhibited the H. pylori-induced apoptosis. Furthermore, a T cell line in which Fas was rendered nonfunctional by a frame shift mutation affecting the cytoplasmic domain was resistant to H. pylori-induced death. The expression of mRNA and protein for FasL on T cell lines was up-regulated by H. pylori and temporally related to H. pylori-mediated killing. Moreover, inhibiting protein synthesis with 10 mM emitine entirely blocked FasL expression and the induction of apoptosis in the T cells after stimulation with H. pylori. Preventing the cleavage of FasL with a metalloproteinase inhibitortl.Itl-phenanthroline)increased H. pylori-mediated killing. Conclusion: H. pylori induces the suicide or fratricide of Pas-bearing T cells through the induction of FasL expression. Thus, persistent infection with strains bearing the cag pathogenicity island may be favored by the induction of local tolerance through the negative selection of T cells encountering H. pylori antigens.