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Interleukin-5-induced thrombopoietin mediates inflammation-associated thrombocytosis
3911
3914
luterleukin-6-1nduced Thrombopoiutin Mediates Inflammation-Associated Thrombocytosis Arthur Kaser, Gerald Brandacher,Wolfgang Steurer, Susanne Kaser, Felix A. Offner, Clemens Molnar, Univ Hosp Innsbruck, Innsbruck Austria; Michael B. Atkins, Beth Israel Deaconess Medical Ctr; James W. Mier, Harvard Medical Sch, Boston, MA; Herbert Tilg, Univ Hosp Innsbruck, Innsbruck Austria
Regulation of Somatostatin and Gautrin by luterferon~ in the Mouse Gastric Mucosa. Yana Zavros, Juanita L. Merchant, Univ of Michigan, Ann Arbor, MI Background: Patients infected with Helicobacter pylori show an increase in gastric mucosal lymphocytesand thus inflammation. Gastric inflammation, associatedwith bacterial infection, results in an increase of IFN~/-expressingT lymphocytes that is indicative of a cell-mediated (Thl) immune response. In addition, infection causes changes in neuroendocdnecell populations including gastrin (G)- and somatostatin(SOM)-secretingD-cells. Proinflammatorycytokines such as IFN'y and TNF~stimulate the release of gastrin and SOM from isolated G- and D-cells respectivelyin vitro. However, in vivo immunoregulation of SOM and gastrin has not been studied in the gastric mucosa. Methods: PBS or IFN~,were infused into C57BL/6 mice at a dose of 250 IU/kg (15 IU/mouse/day)for 2, 5 and 7 days. Plasmagastrin concentrations and tissue SUM content were measured by radioimmunoassay.Tissue sections that include both the fundic and antral regions were fixed for histology and morphometric analysis after immunostained for SOM and gastdn. Results: Within 2 days of infusion, tissue SOM content increasedabovethe PBScontrols in both the fundus (9-fold) and antrum (3-fold) and correlated with an increase in the number of D-cells. There was a reciprocal 2-fold decrease in plasma gastrin concentration with no change in G cells. After a 5 and 7 day infusion, tissue SOM content decreasedby 50% in both fundus and antrum; whereas the number ol G-cells and plasma gastdn concentration increased3-fold. Histologic evaluation revealedthe recruitment of lympbocytes and leukocytes in the gastric mucoea after 2, 5 and 7 days of infusion. Further, staining also revealedthe presenceof SOM-secratingmacrophagesat all time points. Conclusions: In the gastric mucoea, SOM and gastrin are regulated by IFN-y. It is likely that the release of IFN~/, consistent with a predominant Thl response contributes to the neuroendocrine changes observed during Helicobacter pylori infection.
Background.lnflammetion in generaland in particular in the gastrointestinaltract is commonly accompanied by thrombocytosis. Thrombocytosis of inflammation is thought to be related to increasedintedeukin-6 (IL-6) levels. Baselineplatelet production is dependenton thrombopoietin (TPO). Methods.Plasmalevels of TPO were determined by ELISA in samples from a human IL-6 clinical trial. TPO mRNA expression,TPO plasma levels, and platelet counts were determined in 057BL/1B mice receiving murine IL-6 intraperitoneally. Furthermore, C57BIJ 10 mice were additionally administered neutralizinganti-TPOantibody. Results.lL-6 treatment results in elevated TPO plasma levels in patients and C57BL/10 mice. IL-6 administration leads to upregulationof hepaticTPO mRNA expressionand elevatedplateletcounts in C57BL/ 10 mice. Neutralizationof TPO results in abrogation of the increase in platelet count in IL6-treated animals. Discussion.Ourdata indicate that thombocytosis in inflammation might be related to upregulation of TPO by the inflammatory mediator IL-6. We propose a novel regulatory pathwayfor megakaryopoiesisin inflammation, which differs from the situation in health, where plasma TPO levels are regulated mainly by platelets themselves.
3912 Recombinant Human Growth Hormone Downregulated mRNA Expression of Acute Phase Proteins Induced by IL-1,8 and IL-6 in Hep G2 Cell Line Xiaowu Wu, David N. Herndon, Steven E. Wolf, Shriners Hospitals for Children, Galveston, TX
3915
BACKGROUND:Previous in vivo studies showed that recombinant human growth hormone (rhGH) exerted inhibitory effects on gene expression of type I acute phase proteins lAPPs). In this study, we investigatedthe effects of rhGH on mRNA expression of APPs induced by recombinant human IL-1/~ and IL-6 (rhlL-1/~ and rhlL-6) in Hep G2 cells. METHODS:Hep G2 cells were grown to confluence in 6-well plates, and then treated with rhlL-1/~ (2ng/ml) and rhlL-6 (2Ong/ml) alone or combined with rhGH (2,~g/ml) for 0.25, 0.5,1, 2, 4,12 hours. Total RNA was extracted from the ceils after treatment, and levels of mRNA expression for ~-l-acid glycoprotein (Type I APP) and ~l-antitrypsin (Type II APP) were measured semiquantitatively by RT-PCR. The products of RT-PCRwere ran on the 1.5% agarose gel, and analyzed by Digital Imaging Analysis System. RESULTS: Expression of mRNA for ~-l-acid glycoprotein lAGP) was increasedand reachedpeak levelsat 1 hour after rhlL-1/~ stimulation, while mRNA expression for
Gastrin and Cyclooxygenaoe-2 (COX-2) Expression in Helicebacter pylori (Hp) Infected Gastric Ulceration Stanislaw J. Konturek, Dept of Physiology, Univ Sch of Medicine, Cracow Poland; Peter C. Konturek, Dept of Medicine, Univ of Erlangen-Nuramberg,Erlangen Germany; Wladyslaw Bielanski, Aiekcandra Duda, Monika Zuchowicz~Elzbieta Karczewska,Oept of Physiology, Univ Sch of Medicine, Cracow Poland; Eckhart G. Hahn, Dept of Medicine, Univ of Erlangen-Nuremberg, EdangenGermany Background: Gastric ulcerations (GU) have been linked to Hp infection that is accompanied by hypergastrinemiaand enhanced generation of prostaglandins (PG), both involved in the pathogenesisof peptic ulcer but no study was undertakento assessthe relationship between the Hp infection and coexpression of gastrin and COX-es, the rate limiting enzymes in the PG production. Aims: This study was designed 1. to determinethe gene expressionof gastrin and its receptors (CCKe-R) at the margin of GU and in gastric mucosa before and after successful Hp eradication; 2. to assess the plasma levels and gastric luminal contents of gastrin before and after Hp eradication and 3. to examinethe mRNA and protein expression of COX-1 and COX-2 as well as the PGE2generation in ulcer margin and gastric mucosa before and after the Hp eradication. Material and Methods: The trial material included 20 GU patient~ and 40 controls. Gene expressions of gaetdn, CCKB-R, COX-1 and CUX-2 were examined using RT-PCRwith/~actin as a reference and employing Western blot for COX-2 expression, while gastrin and PGE2were measured by RIA. All GUs were located within the antral mucosa. Results: The seroprevalence of Hp and CagA was significantly higher in GU(85%) than in controls (62.5%). Both gastdn and CCKe-RmRNA were detected by RTPCR in ulcer margin and gastrin mRNA was overexprassedin remaining antral mucosa, while CCKB-RmRNAwas overaxpressedin fundic mucosa. Similarly COX-2 mRNA and protein were found in margin of GU and in the Hp infected antral and fundic mucosa but not in the mucosa of Hp eradicated patients in whom ulcers completely healed and gastrin was detected only in antrum, CCI~-R only in corpus, while COX-1 - both in antrum and corpus. Hp positive GUs showed about 2-3 times higher levels of plasma immunoreactive gastrin and PGEzand about 50% higher luminal gastrin contents than the Hp negative controls and this increased plasma and luminal gastdn and tissue PGE2generation were normalized following the Hp eradication. A significant fall in gastrin and CCKB-RmRNA expressionwas noticed six weeks after Hp eradication in gastric antral and fundic mucosa,while COX-2 mRNA completely disappearedafter this treatment. Conclusions: 1) Hp infected GU margin coexpressesgastdn, its receptorsand CUX-2;2. Hp infection may contribute to GU via increased releaseof gastrin and PGE2,and 3. Gastrin produced in Hp infected antral mucosa seems to play a crucial role in the induction of COX-2 and PG generation at GU margin.
Fig. 2 GHand IL-lp Stimulation
lOO,
20
• IL-6 4* IL-6 + GH
•~ ,: .~~ a . . . . . . 0,25 0.5 1 2 4 12 Time (Hour)
20
• •
IL-I~ IL-11~+ GH
lo," -- cont~ . 0,25 0.5 t 2 4 "Nine(Hour)
12
3913 Gastrin Induces IL-8 Expression in Gastric Epithelial Cells Through the Activation of NF-kappaB and AP-1 Yoshiji Miyazaki,Sbintaro Hiraoka, Shinji Kitamura, Miyuki Toyota, Tatsuya Kiyohara, Yutaka Nagasawa,Yasuhisa Shinomura, Yuji Matsuzawa,GraduateSch of Medicine, Osaka Univ, OsakaJapan Background and Aims: Hypergastrinemiais frequently observed in individuals with a chronic Helicobacterpylori infection. However,the pathophysiologicalsignificance of hypergastrinemia in gastric mucosal inflammation is unclear. The present study was designed to determine if gastrin induces the expression of IL-8 in human and rat gastric epithelial cells. Methods: Human and rat gastric epithelial cells, transfected with gastdn receptor, were stimulated with gastrin. The expression of IL-8 mRNA and release of its protein were then determined by Northern blot analysis and an enzyme-linked immunosorbent assay, respectively. Results: Gastrin induced the expressionof IL-8 transcripts and protein and this effect was synergistic with IL-1/~or TNF-e. A luciferase assay using IL-8 promoter genes showed that NF-KB is absolutely required and AP-1 is partly required for the maximum induction of IL-6 by gastrin. An electrophoraUcmobility shift assay revealedthat gastrin is capable of activating both NFKB and AP-1, and that the inhibition of NF-KB abrogated the gastdn induction of IL-B. The inhibition of either protein kinase C or mitogen-activated protein kinase kinase partially suppressed gastdn-induced IL-8 expression.Conclusions: These results suggest that gaetrin is capableof upregulatingI L-8 expressionin gastric epithelialcells and thereforemay contribute to the progression of the inflammatory process in the stomach.
3916 Changes in Gastric Mucosal Perexisome Praliferator-Activated Receptor ,y (PPAR~,) Expression After Helicobacler pylori Eradication Atsushi Nakajima, Emi Oeawa, Hisahiko Sekihara, Yokohama City Univ, YokohamaJapan; Sachiyo Nomura, Michio Kaminishi, Nobuyuki Metsuhashi, Univ of Tokyo, Tokyo Japan PURPOSE: Expression of peroxisome proliferator-activated receptor .y (PPAR-,/) has been demonstrated in some gastric cancer cells. Although PPAR~/ligandsare reportedto suppress cell proliferation in gastric cancer cell lines, the precise role of PPAR~,expressedin the gastric cancer cells is unknown. Since chronic atrophic gastritis, hyperplastic polyp, gastric adenoma and gastric cancer are all known to be associated with chronic Helicobacter pylori (Hp) infection, we investigatedgastric mucosalexpressionof PPAR~/beforeand after Hperedication to elucidatethe role of the receptorin those settings. METHODS:Hp eradicationwas performed in 9 patientswith chronic atrophic gastritis, 5 patients with gastric adenomas,and 10 patients with hyperplasUcpolyps. All patients were positive for Hp. Biopsy samples of the lesions obtained before and after the Hp eradication were subjected to immunohistochemistry for PPAR~t.All hyperplastic polyps regressedafter Hp eradication, while adenomasshowed little or no regression. RESULTS:Prior to Hp eradication,all samples of chronic atrophic gastritis and hyperplaeticpolyps were positive for PPAR-t, especially in infiltrating inflammatory cells.
A-727
3914
luterleukin-6-1nduced Thrombopoiutin Mediates Inflammation-Associated Thrombocytosis Arthur Kaser, Gerald Brandacher,Wolfgang Steurer, Susanne Kaser, Felix A. Offner, Clemens Molnar, Univ Hosp Innsbruck, Innsbruck Austria; Michael B. Atkins, Beth Israel Deaconess Medical Ctr; James W. Mier, Harvard Medical Sch, Boston, MA; Herbert Tilg, Univ Hosp Innsbruck, Innsbruck Austria
Regulation of Somatostatin and Gautrin by luterferon~ in the Mouse Gastric Mucosa. Yana Zavros, Juanita L. Merchant, Univ of Michigan, Ann Arbor, MI Background: Patients infected with Helicobacter pylori show an increase in gastric mucosal lymphocytesand thus inflammation. Gastric inflammation, associatedwith bacterial infection, results in an increase of IFN~/-expressingT lymphocytes that is indicative of a cell-mediated (Thl) immune response. In addition, infection causes changes in neuroendocdnecell populations including gastrin (G)- and somatostatin(SOM)-secretingD-cells. Proinflammatorycytokines such as IFN'y and TNF~stimulate the release of gastrin and SOM from isolated G- and D-cells respectivelyin vitro. However, in vivo immunoregulation of SOM and gastrin has not been studied in the gastric mucosa. Methods: PBS or IFN~,were infused into C57BL/6 mice at a dose of 250 IU/kg (15 IU/mouse/day)for 2, 5 and 7 days. Plasmagastrin concentrations and tissue SUM content were measured by radioimmunoassay.Tissue sections that include both the fundic and antral regions were fixed for histology and morphometric analysis after immunostained for SOM and gastdn. Results: Within 2 days of infusion, tissue SOM content increasedabovethe PBScontrols in both the fundus (9-fold) and antrum (3-fold) and correlated with an increase in the number of D-cells. There was a reciprocal 2-fold decrease in plasma gastrin concentration with no change in G cells. After a 5 and 7 day infusion, tissue SOM content decreasedby 50% in both fundus and antrum; whereas the number ol G-cells and plasma gastdn concentration increased3-fold. Histologic evaluation revealedthe recruitment of lympbocytes and leukocytes in the gastric mucoea after 2, 5 and 7 days of infusion. Further, staining also revealedthe presenceof SOM-secratingmacrophagesat all time points. Conclusions: In the gastric mucoea, SOM and gastrin are regulated by IFN-y. It is likely that the release of IFN~/, consistent with a predominant Thl response contributes to the neuroendocrine changes observed during Helicobacter pylori infection.
Background.lnflammetion in generaland in particular in the gastrointestinaltract is commonly accompanied by thrombocytosis. Thrombocytosis of inflammation is thought to be related to increasedintedeukin-6 (IL-6) levels. Baselineplatelet production is dependenton thrombopoietin (TPO). Methods.Plasmalevels of TPO were determined by ELISA in samples from a human IL-6 clinical trial. TPO mRNA expression,TPO plasma levels, and platelet counts were determined in 057BL/1B mice receiving murine IL-6 intraperitoneally. Furthermore, C57BIJ 10 mice were additionally administered neutralizinganti-TPOantibody. Results.lL-6 treatment results in elevated TPO plasma levels in patients and C57BL/10 mice. IL-6 administration leads to upregulationof hepaticTPO mRNA expressionand elevatedplateletcounts in C57BL/ 10 mice. Neutralizationof TPO results in abrogation of the increase in platelet count in IL6-treated animals. Discussion.Ourdata indicate that thombocytosis in inflammation might be related to upregulation of TPO by the inflammatory mediator IL-6. We propose a novel regulatory pathwayfor megakaryopoiesisin inflammation, which differs from the situation in health, where plasma TPO levels are regulated mainly by platelets themselves.
3912 Recombinant Human Growth Hormone Downregulated mRNA Expression of Acute Phase Proteins Induced by IL-1,8 and IL-6 in Hep G2 Cell Line Xiaowu Wu, David N. Herndon, Steven E. Wolf, Shriners Hospitals for Children, Galveston, TX
3915
BACKGROUND:Previous in vivo studies showed that recombinant human growth hormone (rhGH) exerted inhibitory effects on gene expression of type I acute phase proteins lAPPs). In this study, we investigatedthe effects of rhGH on mRNA expression of APPs induced by recombinant human IL-1/~ and IL-6 (rhlL-1/~ and rhlL-6) in Hep G2 cells. METHODS:Hep G2 cells were grown to confluence in 6-well plates, and then treated with rhlL-1/~ (2ng/ml) and rhlL-6 (2Ong/ml) alone or combined with rhGH (2,~g/ml) for 0.25, 0.5,1, 2, 4,12 hours. Total RNA was extracted from the ceils after treatment, and levels of mRNA expression for ~-l-acid glycoprotein (Type I APP) and ~l-antitrypsin (Type II APP) were measured semiquantitatively by RT-PCR. The products of RT-PCRwere ran on the 1.5% agarose gel, and analyzed by Digital Imaging Analysis System. RESULTS: Expression of mRNA for ~-l-acid glycoprotein lAGP) was increasedand reachedpeak levelsat 1 hour after rhlL-1/~ stimulation, while mRNA expression for
Gastrin and Cyclooxygenaoe-2 (COX-2) Expression in Helicebacter pylori (Hp) Infected Gastric Ulceration Stanislaw J. Konturek, Dept of Physiology, Univ Sch of Medicine, Cracow Poland; Peter C. Konturek, Dept of Medicine, Univ of Erlangen-Nuramberg,Erlangen Germany; Wladyslaw Bielanski, Aiekcandra Duda, Monika Zuchowicz~Elzbieta Karczewska,Oept of Physiology, Univ Sch of Medicine, Cracow Poland; Eckhart G. Hahn, Dept of Medicine, Univ of Erlangen-Nuremberg, EdangenGermany Background: Gastric ulcerations (GU) have been linked to Hp infection that is accompanied by hypergastrinemiaand enhanced generation of prostaglandins (PG), both involved in the pathogenesisof peptic ulcer but no study was undertakento assessthe relationship between the Hp infection and coexpression of gastrin and COX-es, the rate limiting enzymes in the PG production. Aims: This study was designed 1. to determinethe gene expressionof gastrin and its receptors (CCKe-R) at the margin of GU and in gastric mucosa before and after successful Hp eradication; 2. to assess the plasma levels and gastric luminal contents of gastrin before and after Hp eradication and 3. to examinethe mRNA and protein expression of COX-1 and COX-2 as well as the PGE2generation in ulcer margin and gastric mucosa before and after the Hp eradication. Material and Methods: The trial material included 20 GU patient~ and 40 controls. Gene expressions of gaetdn, CCKB-R, COX-1 and CUX-2 were examined using RT-PCRwith/~actin as a reference and employing Western blot for COX-2 expression, while gastrin and PGE2were measured by RIA. All GUs were located within the antral mucosa. Results: The seroprevalence of Hp and CagA was significantly higher in GU(85%) than in controls (62.5%). Both gastdn and CCKe-RmRNA were detected by RTPCR in ulcer margin and gastrin mRNA was overexprassedin remaining antral mucosa, while CCKB-RmRNAwas overaxpressedin fundic mucosa. Similarly COX-2 mRNA and protein were found in margin of GU and in the Hp infected antral and fundic mucosa but not in the mucosa of Hp eradicated patients in whom ulcers completely healed and gastrin was detected only in antrum, CCI~-R only in corpus, while COX-1 - both in antrum and corpus. Hp positive GUs showed about 2-3 times higher levels of plasma immunoreactive gastrin and PGEzand about 50% higher luminal gastrin contents than the Hp negative controls and this increased plasma and luminal gastdn and tissue PGE2generation were normalized following the Hp eradication. A significant fall in gastrin and CCKB-RmRNA expressionwas noticed six weeks after Hp eradication in gastric antral and fundic mucosa,while COX-2 mRNA completely disappearedafter this treatment. Conclusions: 1) Hp infected GU margin coexpressesgastdn, its receptorsand CUX-2;2. Hp infection may contribute to GU via increased releaseof gastrin and PGE2,and 3. Gastrin produced in Hp infected antral mucosa seems to play a crucial role in the induction of COX-2 and PG generation at GU margin.
Fig. 2 GHand IL-lp Stimulation
lOO,
20
• IL-6 4* IL-6 + GH
•~ ,: .~~ a . . . . . . 0,25 0.5 1 2 4 12 Time (Hour)
20
• •
IL-I~ IL-11~+ GH
lo," -- cont~ . 0,25 0.5 t 2 4 "Nine(Hour)
12
3913 Gastrin Induces IL-8 Expression in Gastric Epithelial Cells Through the Activation of NF-kappaB and AP-1 Yoshiji Miyazaki,Sbintaro Hiraoka, Shinji Kitamura, Miyuki Toyota, Tatsuya Kiyohara, Yutaka Nagasawa,Yasuhisa Shinomura, Yuji Matsuzawa,GraduateSch of Medicine, Osaka Univ, OsakaJapan Background and Aims: Hypergastrinemiais frequently observed in individuals with a chronic Helicobacterpylori infection. However,the pathophysiologicalsignificance of hypergastrinemia in gastric mucosal inflammation is unclear. The present study was designed to determine if gastrin induces the expression of IL-8 in human and rat gastric epithelial cells. Methods: Human and rat gastric epithelial cells, transfected with gastdn receptor, were stimulated with gastrin. The expression of IL-8 mRNA and release of its protein were then determined by Northern blot analysis and an enzyme-linked immunosorbent assay, respectively. Results: Gastrin induced the expressionof IL-8 transcripts and protein and this effect was synergistic with IL-1/~or TNF-e. A luciferase assay using IL-8 promoter genes showed that NF-KB is absolutely required and AP-1 is partly required for the maximum induction of IL-6 by gastrin. An electrophoraUcmobility shift assay revealedthat gastrin is capable of activating both NFKB and AP-1, and that the inhibition of NF-KB abrogated the gastdn induction of IL-B. The inhibition of either protein kinase C or mitogen-activated protein kinase kinase partially suppressed gastdn-induced IL-8 expression.Conclusions: These results suggest that gaetrin is capableof upregulatingI L-8 expressionin gastric epithelialcells and thereforemay contribute to the progression of the inflammatory process in the stomach.
3916 Changes in Gastric Mucosal Perexisome Praliferator-Activated Receptor ,y (PPAR~,) Expression After Helicobacler pylori Eradication Atsushi Nakajima, Emi Oeawa, Hisahiko Sekihara, Yokohama City Univ, YokohamaJapan; Sachiyo Nomura, Michio Kaminishi, Nobuyuki Metsuhashi, Univ of Tokyo, Tokyo Japan PURPOSE: Expression of peroxisome proliferator-activated receptor .y (PPAR-,/) has been demonstrated in some gastric cancer cells. Although PPAR~/ligandsare reportedto suppress cell proliferation in gastric cancer cell lines, the precise role of PPAR~,expressedin the gastric cancer cells is unknown. Since chronic atrophic gastritis, hyperplastic polyp, gastric adenoma and gastric cancer are all known to be associated with chronic Helicobacter pylori (Hp) infection, we investigatedgastric mucosalexpressionof PPAR~/beforeand after Hperedication to elucidatethe role of the receptorin those settings. METHODS:Hp eradicationwas performed in 9 patientswith chronic atrophic gastritis, 5 patients with gastric adenomas,and 10 patients with hyperplasUcpolyps. All patients were positive for Hp. Biopsy samples of the lesions obtained before and after the Hp eradication were subjected to immunohistochemistry for PPAR~t.All hyperplastic polyps regressedafter Hp eradication, while adenomasshowed little or no regression. RESULTS:Prior to Hp eradication,all samples of chronic atrophic gastritis and hyperplaeticpolyps were positive for PPAR-t, especially in infiltrating inflammatory cells.
A-727