453
The Lipocortin of Colon Mucosa in Ulcerative Colitis Y. Sakanoue, M. Kusunoki, T. Hatada, T. Sakiyama, S. Fujita, T. Yamamura and J. Utsunomiya Second Department of Surgery, Hyogo College of Medicine, Hyogo, Japan
When the inflammatory response becomes prolonged in duration or excessive in its magnitude — chronic inflammatory bowel disease (ulcerative colitis) may be an example of this — it is at this point that the clinician and surgeon step in to administer some form of therapy. However, the mechanisms responsible for the initiation of inflammation, for terminating the response, and for turning the inflammation into chronic state in some clinical setting are not well understood. Lipocortins are a family of anti-inflammatory proteins acting by inhibiting the activity of phospholipase A2, and thus preventing the formation of inflammatory mediators, prostaglandins and leukotrienes (DiRosa, Flower, Hirata, Parente and Russo-Marie Fig. 1 Western blot analyses of lipocortin in normal and inflamed 1984). The aim of this study is to know the amounts of lipocortins of colonic mucosa, normal colon mucosa obtained from patient with colonic mucosa in ulcerative colitis.
hereditary nonpolyposis colon cancer and inflamed colon mucosa obtained from patient with ulcerative colitis. The homogenates were Materials and Methods boiled in a Laemmli sample buffer for 5 min. The samples were then processed for immunoblots with antibodies to lipocortin as deMaterials scribed in Materials and Methods. Lipocortin II (calpactin 1, heravy chain) was Track 1 and 2, normal colon mucosa homogenates (transverse colon purified from bovine lung (Shaddle, Gerke and Weber 1985). A poly- 1, sigmoid colon 2) and track 3 and 4, inflamed colon mucosa homoclonal antibody against purified lipocortin was prepared and detected genates (transverse colon 3, sigmoid colon 4), track 5, molecular by ELISA (Ek andHeldin1984). weight marker proteins.
Tissue specimens Inflamed colonic specimens were obtained at total colectomy from 5 patients with ulcerative colitis (3 male and 2 female; ranging from 24 yr to 40 yr). The diagnosis was based on clinical and pathological criteria, and endoscopical disease activity was severe. These patients were treated with corticosteroids (prednisolone 20 mg) before operative day. Histologically normal colon mucosa were obtained at total colectomy from 40 years old male patient with hereditary nonpolyposis ascending colon cancer. Other normal colonic samples were one rectal cancer (52 yr, male) and two sigmoid cancer (48 yr male, 56 yr female). All specimens were washed throughly and collected in icecold phosphate-buffered saline. The tissues were cut into small pieces and homogenized in 5 vol of buffer A (10 mM Hepes-NaOH pH 7.6, 5 mM EDTA, 100 mM NaCl 200 µM PMSF, 1000 U/ml aprotinin) using a polytron homogenizer.
Immune blot analysis After adjusting the protein concentration equally in each pair of experiments, an aliquot of each extract (100µgof protein) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis {Laemmli 1970).
Horm. meta. Res. 22 (1990) 453 - 454 © Georg Thieme Verlag Stuttgart • New York
Electrophoretic transfer onto nitrocellulose sheets (western blot analysis) was performed (Towbin, Staehelin and Gordon 1979) and antigenic bands were detected by the Immun-blot assay using the rabbit-anti lipocortin antibody (1:200 dilution) followed by Protein A conjugated to horseradish peroxidase and horseradish peroxidase substrate (as described in the Immun-blot Assay Kit, BioRad Laboratories). Results As shown in Figure 1, a polyclonal antibody against lipocortin was found on western blot analysis to react with lipocortin (36000-Mr) of normal colon mucosa (transverse colon, track 1 and sigmoid colon, track 2). In contrast inflamed colon mucosa obtained from a patient with ulcerative colitis did not react with the antilipocortin antibody on western blots (transverse colon, track 3 and sigmoid colon, track 4), even when large amounts (up to 200 ug protein were applied to the gel) of material adapted. Inflamed colon mucosa obtained from other patients had been observed with the same results in the pattern of immune blot analysis. A control antibody was ineffective in the same conditions (data not shown). In addition, there is no difference in the amounts of lipocortin in normal colon mucosa between right sided colon and left sided colon mucosa.
Received: 14 Nov. 1989
Accepted: 3 May 1990 after revision
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Introduction
Y. Sakanoue, M. Kusunoki, T. Hatada, T. Sakiyama, S. Fujita, T. Yamamura, J. Utsunomiya
Discussion The results of this preliminary study show that significant differences exist among the amounts of lipocortin in human colon mucosa. When compared with normal colon mucosa in four patients with carcinoma, lipocortin could be detected using immuno blot analysis, whereas inflamed colon mucosa from five patients with ulcerative colitis did not contain lipocortin.
Acknowledgements We thank Ms. E. Matsuno and Ms. T. Okada for their technical and secretarial assistance.
References
DiRosa, M., R. J. Flower, F. Hirata, L. Parente, F. Russo-Marie: Prostaglandins 28: 441-444(1984) This observation suggests that lipocortin was not preEk, B., C.H.Heldin:J. Biol. Chem. 259:11145-11152 (1984) sent in colon mucosa from a patient with ulcerative colitis, therefore, Flower, R. J., L. Parente, P. Persico, J. A. Solmon: Br. J. Pharmacol. the inflammatory reaction would become greatly exaggerated in re(1985) sponse to a given stimulus. In fact Flower, Parente, Persico and Solmon Huebner, K., L. A. Connizzaro, A. Z. Frey, B. K. Hechet, F. Hechet, C. (1985) reported that adrenalectomized rats respond to a standard dose Croce, B. P. Wallner.Oaco. Res. 2:299-310 (1988) of carageenin with a greatly exaggerated inflammatory response and Laemmli, U./^..Nature 227:680-685(1970) enhanced mediator production as compared with sham-operated conShaddle, P. J., V. Gerke, K. Weber:}. Biol. Chem. 260: 16354-16366 trol rats. However, it cannot be denied that this observation results in (1985) an inflammatory change of colonic mucosa. A patient with ulcerative Towbin, H., T. Taehelin, J. Gordon: Proc. Natl. Acad. Sci. USA 76: colitis was on prednisolone which was needed to enable remission to 4350-4354(1979) be induced. Pharmacological effects of this drug on colonic lipocortin at the step of induction of lipocortin are not clear. Requests for reprints should be addressed to: Recently the cDNA clones for two members of the Youichirou Sakanoue, M. D. lipocortin family, lipocortin I and II, have been isolated and their sequences determined to be 50% homologous similar biochemical. Second Department of Surgery properties and structural features of the lipocortins have established in Hyogo College of Medicine various cells and tissues {Huebner, Connizzaro, Frey, Hechet, Hechet, 1-1 Mukogawa-cho, Nishinomiya Croce and Wallner 1988). We have carried out immuno blot analysis Hyogo 663 (Japan) against lipocortin II. These observations have focused attention on the lipocortins and their role in controlling defense reactions against inflammation.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
454 Horm. meta. Res. 22 (1990)
The Lipocortin of Colon Mucosa in Ulcerative Colitis Y. Sakanoue, M. Kusunoki, T. Hatada, T. Sakiyama, S. Fujita, T. Yamamura and J. Utsunomiya Second Department of Surgery, Hyogo College of Medicine, Hyogo, Japan
When the inflammatory response becomes prolonged in duration or excessive in its magnitude — chronic inflammatory bowel disease (ulcerative colitis) may be an example of this — it is at this point that the clinician and surgeon step in to administer some form of therapy. However, the mechanisms responsible for the initiation of inflammation, for terminating the response, and for turning the inflammation into chronic state in some clinical setting are not well understood. Lipocortins are a family of anti-inflammatory proteins acting by inhibiting the activity of phospholipase A2, and thus preventing the formation of inflammatory mediators, prostaglandins and leukotrienes (DiRosa, Flower, Hirata, Parente and Russo-Marie Fig. 1 Western blot analyses of lipocortin in normal and inflamed 1984). The aim of this study is to know the amounts of lipocortins of colonic mucosa, normal colon mucosa obtained from patient with colonic mucosa in ulcerative colitis.
hereditary nonpolyposis colon cancer and inflamed colon mucosa obtained from patient with ulcerative colitis. The homogenates were Materials and Methods boiled in a Laemmli sample buffer for 5 min. The samples were then processed for immunoblots with antibodies to lipocortin as deMaterials scribed in Materials and Methods. Lipocortin II (calpactin 1, heravy chain) was Track 1 and 2, normal colon mucosa homogenates (transverse colon purified from bovine lung (Shaddle, Gerke and Weber 1985). A poly- 1, sigmoid colon 2) and track 3 and 4, inflamed colon mucosa homoclonal antibody against purified lipocortin was prepared and detected genates (transverse colon 3, sigmoid colon 4), track 5, molecular by ELISA (Ek andHeldin1984). weight marker proteins.
Tissue specimens Inflamed colonic specimens were obtained at total colectomy from 5 patients with ulcerative colitis (3 male and 2 female; ranging from 24 yr to 40 yr). The diagnosis was based on clinical and pathological criteria, and endoscopical disease activity was severe. These patients were treated with corticosteroids (prednisolone 20 mg) before operative day. Histologically normal colon mucosa were obtained at total colectomy from 40 years old male patient with hereditary nonpolyposis ascending colon cancer. Other normal colonic samples were one rectal cancer (52 yr, male) and two sigmoid cancer (48 yr male, 56 yr female). All specimens were washed throughly and collected in icecold phosphate-buffered saline. The tissues were cut into small pieces and homogenized in 5 vol of buffer A (10 mM Hepes-NaOH pH 7.6, 5 mM EDTA, 100 mM NaCl 200 µM PMSF, 1000 U/ml aprotinin) using a polytron homogenizer.
Immune blot analysis After adjusting the protein concentration equally in each pair of experiments, an aliquot of each extract (100µgof protein) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis {Laemmli 1970).
Horm. meta. Res. 22 (1990) 453 - 454 © Georg Thieme Verlag Stuttgart • New York
Electrophoretic transfer onto nitrocellulose sheets (western blot analysis) was performed (Towbin, Staehelin and Gordon 1979) and antigenic bands were detected by the Immun-blot assay using the rabbit-anti lipocortin antibody (1:200 dilution) followed by Protein A conjugated to horseradish peroxidase and horseradish peroxidase substrate (as described in the Immun-blot Assay Kit, BioRad Laboratories). Results As shown in Figure 1, a polyclonal antibody against lipocortin was found on western blot analysis to react with lipocortin (36000-Mr) of normal colon mucosa (transverse colon, track 1 and sigmoid colon, track 2). In contrast inflamed colon mucosa obtained from a patient with ulcerative colitis did not react with the antilipocortin antibody on western blots (transverse colon, track 3 and sigmoid colon, track 4), even when large amounts (up to 200 ug protein were applied to the gel) of material adapted. Inflamed colon mucosa obtained from other patients had been observed with the same results in the pattern of immune blot analysis. A control antibody was ineffective in the same conditions (data not shown). In addition, there is no difference in the amounts of lipocortin in normal colon mucosa between right sided colon and left sided colon mucosa.
Received: 14 Nov. 1989
Accepted: 3 May 1990 after revision
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Introduction
Y. Sakanoue, M. Kusunoki, T. Hatada, T. Sakiyama, S. Fujita, T. Yamamura, J. Utsunomiya
Discussion The results of this preliminary study show that significant differences exist among the amounts of lipocortin in human colon mucosa. When compared with normal colon mucosa in four patients with carcinoma, lipocortin could be detected using immuno blot analysis, whereas inflamed colon mucosa from five patients with ulcerative colitis did not contain lipocortin.
Acknowledgements We thank Ms. E. Matsuno and Ms. T. Okada for their technical and secretarial assistance.
References
DiRosa, M., R. J. Flower, F. Hirata, L. Parente, F. Russo-Marie: Prostaglandins 28: 441-444(1984) This observation suggests that lipocortin was not preEk, B., C.H.Heldin:J. Biol. Chem. 259:11145-11152 (1984) sent in colon mucosa from a patient with ulcerative colitis, therefore, Flower, R. J., L. Parente, P. Persico, J. A. Solmon: Br. J. Pharmacol. the inflammatory reaction would become greatly exaggerated in re(1985) sponse to a given stimulus. In fact Flower, Parente, Persico and Solmon Huebner, K., L. A. Connizzaro, A. Z. Frey, B. K. Hechet, F. Hechet, C. (1985) reported that adrenalectomized rats respond to a standard dose Croce, B. P. Wallner.Oaco. Res. 2:299-310 (1988) of carageenin with a greatly exaggerated inflammatory response and Laemmli, U./^..Nature 227:680-685(1970) enhanced mediator production as compared with sham-operated conShaddle, P. J., V. Gerke, K. Weber:}. Biol. Chem. 260: 16354-16366 trol rats. However, it cannot be denied that this observation results in (1985) an inflammatory change of colonic mucosa. A patient with ulcerative Towbin, H., T. Taehelin, J. Gordon: Proc. Natl. Acad. Sci. USA 76: colitis was on prednisolone which was needed to enable remission to 4350-4354(1979) be induced. Pharmacological effects of this drug on colonic lipocortin at the step of induction of lipocortin are not clear. Requests for reprints should be addressed to: Recently the cDNA clones for two members of the Youichirou Sakanoue, M. D. lipocortin family, lipocortin I and II, have been isolated and their sequences determined to be 50% homologous similar biochemical. Second Department of Surgery properties and structural features of the lipocortins have established in Hyogo College of Medicine various cells and tissues {Huebner, Connizzaro, Frey, Hechet, Hechet, 1-1 Mukogawa-cho, Nishinomiya Croce and Wallner 1988). We have carried out immuno blot analysis Hyogo 663 (Japan) against lipocortin II. These observations have focused attention on the lipocortins and their role in controlling defense reactions against inflammation.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
454 Horm. meta. Res. 22 (1990)
