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The possible role of mouse intestinal epithelial cells in the stimulation of lamina propria lymphocytes
April 1995
• EXPRESSION OF THE CD8 LIGAND, gpl80, BY EPITHELIAL CELLS FROM PATIENTS WITH IBD. X.Y. Yio, L.S. Toy, A.Y. Lin, S. Honig, L. Mayer. Division of Clinical Immunology, Mount Sinai Medical Center, New York, New York 10029. We have previously reported the existence of an epithelial cell specific surface molecule gpl80 which binds to CD8 and activates CD8 associated p561ck. This molecule, recognized by the mAb B9, appears to be important in the activation of CD8 + T cells by normal IECs. Since IEC derived from patients with IBD fail to stimulate CD8 + T cells, we sought tO determine whether gplS0 expression was altered in these patients by both immuno-histochemical and flow cytometric analyses, mAb B9 staining of frozen sections of normal bowel revealed bright staining on all epithelial cells (villus > crypt) with a more apical distribution. In contrast, in UC, epithelial cell staining was more patchy with areas of nonstaining in both inflamed as well as noninflamed areas. In CD, staining was absent in many specimens with only faint staining in o t h e r s . These data were confirmed in a more quantitative fashion by flow cytometry. IEC were isolated from surgical specimens or biopsies by dispase treatment, stained with FITC conjugated mAb B9 and anti-class I (W6/32). gplS0 expression was first analyzed in normal IEC derived from multiple sites in the colon and small bowel. While there was some variability in various bowel segments there was always >95% staining and fluorescence intensity was comparable regardless of the region sampled. IEC derived from patients with UC exhibited two populations, staining and nonstaining (40-80%), with a fluorescence intensity in the staining population comparable to normal IEC: IEC derived from patients with CD demonstrated either no staining or very weak staining with mAb B9. Thus gpl80 expression by IBD IEC appears to be altered and may be a critical component in the lack of CD8 + T cell activation seen.
THE POSSIBLE ROLE OF MOUSE INTESTINAL EPITHELIAL CELLS IN THE STIMULATION OF L A M I N A PROPRIA LYMPHOCYTES. N. Yoshizaki, K.V. Kesari, K.M. Das. UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. Intestinal epithelial cells (iEe) express class II antigens of the major histocompatibility complex (MHC) and can function as antigen presenting cells (APC). The iEC, in contrast to the conventional APC, are known to activate T cells of the suppressor phenotype in the mixed cell culture with peripheral blood lymphocytes (PBL). It has been reported recently that human iEC are capable of specifically stimulating allogeneic lamina propria T cells (LFT), which possess helper activity, in an MHe-independent manner. Interestingly, LPT cells do not appear to respond efficiently to the conventional APC. Thus, the iEC and LPT offer a system to explore new pathways of antigen presentation and T cell stimulation. To extend the observations made using human cells and to elucidate the antigenic requirements for the interaction between iEC and LPT cells, we have used genetically-defined inbred strains Of mice. The APC, either splenic or iEC from colon, were obtained from Balb/c (H-2d) mice. The responder cells, either splenic or LFT, were obtained from C3E (H-2 ~) mice. The mixed cell cultures were incubated for 120 h and the stimulation was measured by 3H-thymidine incorporation during the last 18 h. Our preliminary data suggest that m o u s e iEC, like their human counterpart, may function a s APC for the LPT cells (2.5 fold stimulation over the unstimulated control). Further, the iEC appear to be better APe for LPT than for splenic lymphocytes. On the contrary, conventional splenic APC do not s e e m to stimulate LPT cells. We have not yet established whether the stimulation of LPT by iEC is MHC-restricted, but the data suggest that at least the minor lymphocyte stimulatinq (mls) antigens are not involved. The known mls loci are identical between the MHC disparate Balb/c and C3H mice. Interestingly, one anticolon monoclonal antibody (mAb), 7EI~u, inhibits the stimulation of LPT cells by iEC (over 95% reduction in the ~H-thymidine incorporation when compared with the isotype control mAb, MOPCl04E). This mAb reacts specifically to both h u m a n a n d murine colonic epithelial cells and not with s m a l l intestinal enterocytes. The identification and characterization of the antigen, recognized by the 7EI2H,~ numb, should allow us to determine at least one immunoregulatory determinant expressed by the iEC.
Immunology, Microbiology, and Inflammatory Disorders A947
"BAMBOO-JOINT LIKE EROSIONS" OF THE GASTRIC BODY AND CARDIA: A NOVEL SIGN AS A CLUE OF HAVING CROHN'S DISEASE. K. Yokota. M. Yuki, T. Ohta, S. Okuyama, Y. Kohgo. Dept. of Internal Medicine (III), Asahikawa Medical College, Asahikawa, Hokkaido 078, Japan. PURPOSE: Crohn's disease involves not only the ileum and colon but also the stomach and proximal duodenum. Although the gastroduodenal lesions of Crohn's disease rarely cause serious problems, such as, gastric outlet obstruction and intractable ulcerations, the milder lesions are well known to involve these areas. Chronic erosions in the gastric antrum are the most commonly encountered change in Crohn's disease patients. However, these erosions are indistinguishable from those of ordinarily encountered erosive gastritis. Since we experienced a case of Crohn's disease patient who showed a cardiac lesion in Aug. 1989, we have paid attention to the lesser curvature of the gastric body and cardia in the endoscopic observation of Crohn's disease patients. Consequently we noted curious macroscopic findings on these areas of the stomach. We will herein present this endoscopic sign. METHODS: A total of 27 cases of Crohn's disease patients underwent gastroscopic examination from Aug. 1989 to Oct. 1994, using a fide-view type video endoscope (Olympus GF200) and dye-spreading procedure. Fifty serial sections were made from biopsy specimens to detect granulomas. The gastric body lesion was found in 14 patients (52%) out of 27 cases of Crohn's disease. This lesion consisted of 2 to 4 enlarged folds transversed by fissuring erosions in a longitudinal alignment on the lesser curvature of the eardia to middle gastric body, but lacking a longitudinal ulcer. We named this endoscopic sign "bamboojoint like erosions." Microgranulomas were detected in 9 cases (64%) out of 14 patients with bamboo joint-like erosions, by examining 50 serial sections of biopsy specimens. To confirm this sign requires a side-view type endoscope, dye-spreading procedure and adjustment (reduction) of intragastrie air, since this is a very delicate sign and this area is difficult to observe with a direct-view type endoscope. The most important thing, however, is to pay attention to these areas. In our experience, the bamboo-joint like erosions of the gastric body are characteristic to Crohn's disease. We propose here the bamboo joint-like erosions of the gastric body as a novel endoscopic sign of Crohn's disease.
• EFFECTS OF VARIOUS DRUGS ON CHRONIC COLITIS INDUCED BY DEXTRAN SULFATE SODIUM (DSS) IN RATS M.Yoshizumi, I.Hirata, S.Sasaki, K.Takada, ILKumano, H.Morikawa, K~Yoshimura, K.Maemura, O.Saitoh, K.Katsu. The 2nd Dept. of Internal Medicine, Osaka Medical College, Osaka, Japan This study was designed to evaluate the effects of the following agents, salazosulfapyridine (SASP), rebamipide (REB), anti-leukotfiene B4 (antiLTB4) and predonisolone (PSL), against chronic colitis induced by DSS (MW 5,000),whichi was an animal model of human ulcerative colitis. MATERIALS: Male SD rats (200-250g) METHOD: DSS group (n=5), SASP group (n=5), REB group (n=5), anti-LTB4 group (n=5) and PSL group (n=5) were given 1% DSS solution ad libitum for 8 weeks. Moreover DSS group, SASP group, REB group, anti-LTB4 group and PSL group were orally administered 3ml of 0.5% carboxylmethylcellulose (CMC), SASP at a dose of 160mg/kg, REB at a dose of 30mg/kg, anti-LTB4 at a dose of 12mg/kg suspended in 3ml of 0.5% CMC and PSL at a dose of 0.gmg/kg resolved in 3ml saline, respectively, for last 2 weeks. Control group (n=5) was given distilled water without DSS. The colons were excised 8 weeks after the administration of DSS. RESULTS: 1) Gross and microscopic changes of colonic mucosa: Redness, erosion and inflammatory cell infiltration of colonic mucosa were more severe in DSS group than in SASP, REB, anti-LTB4 and PSL groups. 2) Group Control DSS PSL SASP REB anti-LTB4
chang of weight [ % ]
Hb [ g/dl ]
MPO activity [ mU/mg ]
IL-8 level [ng/mg]
109.t_+1.5 14.08___1.96 0.560±0.225 0.120_+0.163 104.7_+1.5** 9.62±2.81 ** 0.694±0.575 2.144___1.299"* 99.7+__1.8** 13.88_+1.17 0.608_+0.232 0.743_+0.905 103.7-+0.8 ** 10.52-+1.63 0.490±0.111 1.966-+1.415 * 102.2+1.6 ** 10.64-+2.96 * 0.540±0.086 2.305_+1.196 ** 98.4±4.1 ** 10.10_+1.89 0.606-+0.140 0.696_+0.131 ** (by Wilcoxn test vs Control group, **p<0.01, *p<0.05)
CONCLUSION: The gross and microscopic findings in these groups suggest that PSL, SASP, REB and anti-LTB4 had suppressive effect on the development of DSS-induced colitis. It is thought that PSL and anti-LTB4 had the inhibitory effect on the increase of IL-8 level.
• EXPRESSION OF THE CD8 LIGAND, gpl80, BY EPITHELIAL CELLS FROM PATIENTS WITH IBD. X.Y. Yio, L.S. Toy, A.Y. Lin, S. Honig, L. Mayer. Division of Clinical Immunology, Mount Sinai Medical Center, New York, New York 10029. We have previously reported the existence of an epithelial cell specific surface molecule gpl80 which binds to CD8 and activates CD8 associated p561ck. This molecule, recognized by the mAb B9, appears to be important in the activation of CD8 + T cells by normal IECs. Since IEC derived from patients with IBD fail to stimulate CD8 + T cells, we sought tO determine whether gplS0 expression was altered in these patients by both immuno-histochemical and flow cytometric analyses, mAb B9 staining of frozen sections of normal bowel revealed bright staining on all epithelial cells (villus > crypt) with a more apical distribution. In contrast, in UC, epithelial cell staining was more patchy with areas of nonstaining in both inflamed as well as noninflamed areas. In CD, staining was absent in many specimens with only faint staining in o t h e r s . These data were confirmed in a more quantitative fashion by flow cytometry. IEC were isolated from surgical specimens or biopsies by dispase treatment, stained with FITC conjugated mAb B9 and anti-class I (W6/32). gplS0 expression was first analyzed in normal IEC derived from multiple sites in the colon and small bowel. While there was some variability in various bowel segments there was always >95% staining and fluorescence intensity was comparable regardless of the region sampled. IEC derived from patients with UC exhibited two populations, staining and nonstaining (40-80%), with a fluorescence intensity in the staining population comparable to normal IEC: IEC derived from patients with CD demonstrated either no staining or very weak staining with mAb B9. Thus gpl80 expression by IBD IEC appears to be altered and may be a critical component in the lack of CD8 + T cell activation seen.
THE POSSIBLE ROLE OF MOUSE INTESTINAL EPITHELIAL CELLS IN THE STIMULATION OF L A M I N A PROPRIA LYMPHOCYTES. N. Yoshizaki, K.V. Kesari, K.M. Das. UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. Intestinal epithelial cells (iEe) express class II antigens of the major histocompatibility complex (MHC) and can function as antigen presenting cells (APC). The iEC, in contrast to the conventional APC, are known to activate T cells of the suppressor phenotype in the mixed cell culture with peripheral blood lymphocytes (PBL). It has been reported recently that human iEC are capable of specifically stimulating allogeneic lamina propria T cells (LFT), which possess helper activity, in an MHe-independent manner. Interestingly, LPT cells do not appear to respond efficiently to the conventional APC. Thus, the iEC and LPT offer a system to explore new pathways of antigen presentation and T cell stimulation. To extend the observations made using human cells and to elucidate the antigenic requirements for the interaction between iEC and LPT cells, we have used genetically-defined inbred strains Of mice. The APC, either splenic or iEC from colon, were obtained from Balb/c (H-2d) mice. The responder cells, either splenic or LFT, were obtained from C3E (H-2 ~) mice. The mixed cell cultures were incubated for 120 h and the stimulation was measured by 3H-thymidine incorporation during the last 18 h. Our preliminary data suggest that m o u s e iEC, like their human counterpart, may function a s APC for the LPT cells (2.5 fold stimulation over the unstimulated control). Further, the iEC appear to be better APe for LPT than for splenic lymphocytes. On the contrary, conventional splenic APC do not s e e m to stimulate LPT cells. We have not yet established whether the stimulation of LPT by iEC is MHC-restricted, but the data suggest that at least the minor lymphocyte stimulatinq (mls) antigens are not involved. The known mls loci are identical between the MHC disparate Balb/c and C3H mice. Interestingly, one anticolon monoclonal antibody (mAb), 7EI~u, inhibits the stimulation of LPT cells by iEC (over 95% reduction in the ~H-thymidine incorporation when compared with the isotype control mAb, MOPCl04E). This mAb reacts specifically to both h u m a n a n d murine colonic epithelial cells and not with s m a l l intestinal enterocytes. The identification and characterization of the antigen, recognized by the 7EI2H,~ numb, should allow us to determine at least one immunoregulatory determinant expressed by the iEC.
Immunology, Microbiology, and Inflammatory Disorders A947
"BAMBOO-JOINT LIKE EROSIONS" OF THE GASTRIC BODY AND CARDIA: A NOVEL SIGN AS A CLUE OF HAVING CROHN'S DISEASE. K. Yokota. M. Yuki, T. Ohta, S. Okuyama, Y. Kohgo. Dept. of Internal Medicine (III), Asahikawa Medical College, Asahikawa, Hokkaido 078, Japan. PURPOSE: Crohn's disease involves not only the ileum and colon but also the stomach and proximal duodenum. Although the gastroduodenal lesions of Crohn's disease rarely cause serious problems, such as, gastric outlet obstruction and intractable ulcerations, the milder lesions are well known to involve these areas. Chronic erosions in the gastric antrum are the most commonly encountered change in Crohn's disease patients. However, these erosions are indistinguishable from those of ordinarily encountered erosive gastritis. Since we experienced a case of Crohn's disease patient who showed a cardiac lesion in Aug. 1989, we have paid attention to the lesser curvature of the gastric body and cardia in the endoscopic observation of Crohn's disease patients. Consequently we noted curious macroscopic findings on these areas of the stomach. We will herein present this endoscopic sign. METHODS: A total of 27 cases of Crohn's disease patients underwent gastroscopic examination from Aug. 1989 to Oct. 1994, using a fide-view type video endoscope (Olympus GF200) and dye-spreading procedure. Fifty serial sections were made from biopsy specimens to detect granulomas. The gastric body lesion was found in 14 patients (52%) out of 27 cases of Crohn's disease. This lesion consisted of 2 to 4 enlarged folds transversed by fissuring erosions in a longitudinal alignment on the lesser curvature of the eardia to middle gastric body, but lacking a longitudinal ulcer. We named this endoscopic sign "bamboojoint like erosions." Microgranulomas were detected in 9 cases (64%) out of 14 patients with bamboo joint-like erosions, by examining 50 serial sections of biopsy specimens. To confirm this sign requires a side-view type endoscope, dye-spreading procedure and adjustment (reduction) of intragastrie air, since this is a very delicate sign and this area is difficult to observe with a direct-view type endoscope. The most important thing, however, is to pay attention to these areas. In our experience, the bamboo-joint like erosions of the gastric body are characteristic to Crohn's disease. We propose here the bamboo joint-like erosions of the gastric body as a novel endoscopic sign of Crohn's disease.
• EFFECTS OF VARIOUS DRUGS ON CHRONIC COLITIS INDUCED BY DEXTRAN SULFATE SODIUM (DSS) IN RATS M.Yoshizumi, I.Hirata, S.Sasaki, K.Takada, ILKumano, H.Morikawa, K~Yoshimura, K.Maemura, O.Saitoh, K.Katsu. The 2nd Dept. of Internal Medicine, Osaka Medical College, Osaka, Japan This study was designed to evaluate the effects of the following agents, salazosulfapyridine (SASP), rebamipide (REB), anti-leukotfiene B4 (antiLTB4) and predonisolone (PSL), against chronic colitis induced by DSS (MW 5,000),whichi was an animal model of human ulcerative colitis. MATERIALS: Male SD rats (200-250g) METHOD: DSS group (n=5), SASP group (n=5), REB group (n=5), anti-LTB4 group (n=5) and PSL group (n=5) were given 1% DSS solution ad libitum for 8 weeks. Moreover DSS group, SASP group, REB group, anti-LTB4 group and PSL group were orally administered 3ml of 0.5% carboxylmethylcellulose (CMC), SASP at a dose of 160mg/kg, REB at a dose of 30mg/kg, anti-LTB4 at a dose of 12mg/kg suspended in 3ml of 0.5% CMC and PSL at a dose of 0.gmg/kg resolved in 3ml saline, respectively, for last 2 weeks. Control group (n=5) was given distilled water without DSS. The colons were excised 8 weeks after the administration of DSS. RESULTS: 1) Gross and microscopic changes of colonic mucosa: Redness, erosion and inflammatory cell infiltration of colonic mucosa were more severe in DSS group than in SASP, REB, anti-LTB4 and PSL groups. 2) Group Control DSS PSL SASP REB anti-LTB4
chang of weight [ % ]
Hb [ g/dl ]
MPO activity [ mU/mg ]
IL-8 level [ng/mg]
109.t_+1.5 14.08___1.96 0.560±0.225 0.120_+0.163 104.7_+1.5** 9.62±2.81 ** 0.694±0.575 2.144___1.299"* 99.7+__1.8** 13.88_+1.17 0.608_+0.232 0.743_+0.905 103.7-+0.8 ** 10.52-+1.63 0.490±0.111 1.966-+1.415 * 102.2+1.6 ** 10.64-+2.96 * 0.540±0.086 2.305_+1.196 ** 98.4±4.1 ** 10.10_+1.89 0.606-+0.140 0.696_+0.131 ** (by Wilcoxn test vs Control group, **p<0.01, *p<0.05)
CONCLUSION: The gross and microscopic findings in these groups suggest that PSL, SASP, REB and anti-LTB4 had suppressive effect on the development of DSS-induced colitis. It is thought that PSL and anti-LTB4 had the inhibitory effect on the increase of IL-8 level.