Endocrine Cells in the Intestinal Metaplasia of Gastric Mucosa Cesare Bordi, MD, and Mariella Ravazzola, PhD
Sections of gastric mucosa removed during surgery for cancer or peptic ulcer and containing regions of intestinal metaplasia were studied by the immunofluorescence technique using several antiserums against intestinal hormones. Endocrine cells such as cells containing somatostatin, glicentin (gut GLI-I), motilin, and probably cholecystokinin were found within metaplastic intestinal epithelium while secretin and neurotensin, which are present in the normal intestinal mucosa, were not detected in metaplastic epithelium. The endocrine-cell population present in the intestinal metaplasia resembles that found in the cryptal region of the normal small intestine, a finding in accordance with the fact that intestinal metaplasia of gastric mucosa usually reproduces structural and histochemical characteristics of small intestinal crypts. (Am J Pathol 96:391-398, 1979)
IN CHRONIC INFLAMMATION of the gastric mucosa, the epithelial lining of the gastric glands can be replaced by an epithelium morphologically similar to that normally seen in the small intestine. This phenomenon has been called intestinal heterotopia,' intestinalization,2 or intestinal metaplasia,3 the latter term and its underlying interpretation being now the most widely accepted. Metaplastic intestinal epithelium of the stomach shows a capacity for lipid absorption 4 and an enzymatic content 6 comparable to those of the intestinal mucosa. Histologically, the metaplastic epithelium contains cells such as absorptive, goblet, undifferentiated, and Paneth cells characteristic of the small intestine.1 The occurrence of endocrine cells within metaplastic intestinal tissue has long been recognized.' Using silver staining, previous workers mentioned the argentaffin or enterochromaffin (EC) cell type,7 9 while recent ultrastructural observations 10-12 described additional endocrine cell types on the basis of the specific morphology of secretion granules. However, in view of the difficulty of identifying unambiguously endocrine cell types on ultrastructural criteria alone, we have reinvestigated this problem with immunofluorescence, employing different antiserums against intestinal hormones. This study resulted in the identification of a characteristic pattern of endocrine cells in the metaplastic epithelium. From the Institute of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland. Supported by Grant No. 3.120.77 from the Swiss National Science Foundation. Accepted for publication March 7, 1979. Address reprint requests to Dr. C. Bordi, Institute of Pathological Anatomy, University of Parma, 1-43100 Parma, Italy.
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Materials and Methods Samples of gastric mucosa were obtained from 13 patients undergoing gastric resection for gastric peptic ulcer (6 cases) or cancer (7 cases). Nine patients were male and 4 female; their age ranged from 41 to 78 years (mean 61.2). In each patient, a strip of gastric mucosa, approximately 2 cm long, was taken from the lesser curvature, from the anterior and from the posterior wall of the oxyntic region, respectively. A fourth strip was taken from the antropyloric region of the lesser curvature, approximately 2 cm above the pylorus. In addition, in 4 patients, a strip of jejunal mucosa was also taken from the transverse section prepared for gastrojejunal anastomosis. The specimens were fixed for 20-24 hours in Bouin's fluid, dehydrated, and embedded in paraffin. Sections of 5 us were stained with hematoxylin-eosin for the identification of metaplastic areas. Successive sections were stained with the silver method of Grimelius 13 to detect most of the endocrine cell types present within the metaplastic epithelium, while other sections were processed for the indirect immunofluorescence technique,"4 with the use of the following antiserums: rabbit antiglicentin (gut GLI-I) (a gift from Dr. A. J. Moody, Copenhagen, Denmark); rabbit antiglucagon 15K (specific for pancreatic glucagon) (a gift from Dr. R. H. Unger, Dallas, Texas); rabbit antibovine pancreatic polypeptide (BPP) (a gift from Dr. R. E. Chance, Indianapolis, Indiana); rabbit antigastrin (a gift from Dr. W. Gepts, Brussels, Belgium); rabbit antisomatostatin and guinea pig antineurotensin (gifts from Dr. S. Ito, Niigata, Japan); rabbit antisecretin and guinea pig antimotilin (gifts from Dr. N. Yanaihara, Shizuoka-Shi, Japan). Most antiserums were used at a 1:50 dilution, except for antimotilin and antisecretin (1: 10), anti-BPP (1 100) and antisomatostatin (1: 200). After incubation with the respective antiserums, the sections were exposed to fluorescein-labeled sheep antirabbit or antiguinea-pig gammaglobulin serums (Pasteur Institute, Paris, France), counterstained with 0.01 % Evans blue, and observed in a Leitz Orthoplan fluorescence microscope equipped with a Ploemopak illuminator. The specificity of the staining reaction was checked in each case by incubating the sections with antiserums previously adsorbed with an excess of their respective antigen. Only immunofluorescence completely abolished by prior incubation of antiserum with the respective antigen was considered in the study. In addition, to discriminate gastrincontaining cells from cholecystokinin (CCK)-containing cells, which appear to stain with most antigastrin serums 15 (including ours), the latter was used before and after adsorption with an excess of CCK octapeptide (Kinevac, kindly donated by Squibb AG, ZUirich, Switzerland).
Results On histologic examination, areas of intestinal metaplasia appeared as surface invaginations lined by an epithelium resembling that of the small intestine. Villous differentiations were not present. Metaplastic areas varied greatly in number and extent from patient to patient and were usually more pronounced in the antropyloric than in the oxyntic region of the
stomach. Endocrine cells were easily identified within metaplastic epithelium by either silver staining or immunofluorescence. Endocrine cells tended to concentrate at the bottom of the invaginations, and they usually showed an elongated shape with a thin apex reaching the tubular lumen (open type). In any sample, the number of endocrine cells varied considerably from one metaplastic region to another even when these were contiguous.
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Also, with any antiserum, areas with fluorescent cells and areas devoid of these cells coexisted in the same patient. The cumulated number of immunofluorescent cells present in a discrete area of the metaplasia was smaller than that of cells stained with Grimelius' silver in the same area, the former usually ranging between 60% and 80% of the latter. Somatostatin
Metaplastic epithelium contained somatostatin-reactive cells in all patients studied, although in highly variable numbers (Figure 1). Moreover, somatostatin-containing cells were always detected in nontransformed areas of the oxyntic and antral mucosa as well as in the normal jejunal epithelium. In antral mucosa these cells were three to four times more numerous than in metaplastic epithelium or in oxyntic and jejunal mucosa. In this latter region fluorescent cells were more frequent in the crypts than in the villi. Glicentin
Positive cells were observed within metaplastic areas of the stomach in all cases except one. Again, the number of glicentin-containing cells was variable, varying from very low (one cell per 3-4 crypts) to very high frequency (numerous cells within the same crypt, as shown in Figure 2). Positive cells were not seen in normal gastric mucosa, but they were seen in jejunal epithelium, especially at the cryptal level. Motilin
Fluorescent cells were found in metaplastic gastric areas (9 out of 13 cases) (Figure 3) and in all jejunal samples studied. In metaplastic epithehum, they were usually fewer than cells containing somatostatin or glicentin. Motilin-containing cells were also found in the uninvolved antropyloric mucosa of 4 patients, but not in oxyntic mucosa. Gastrin-CCK
Cells reacting with antigastrin antiserum were found in metaplastic areas (9 out of 13 cases) as well as in uninvolved antropyloric and in jejunal mucosa (all cases). In the uninvolved antropyloric mucosa these cells were localized in the mid-portion of the glands, and they showed the characteristic round or triangular shape reported for normal antral G cells.'6 On the other hand, fluorescent cells of metaplastic and jejunal epithelium usually appeared more elongated and were located preferentially in the lower part of the crypts (Figure 4A). Adsorption of antigastrin antiserum with the CCK octapeptide did not affect thc immuno-
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fluorescent reaction in cells of the uninvolved antropyloric region but completely abolished that of cells localized in metaplastic or jejunal epithelium (Figure 4B) (except for one case, in which approximately one in every six cells remained fluorescent in the metaplastic epithelium following incubation with the adsorbed antiserum). Secretin
No positive cells were found within metaplastic zones of the stomach, nor in nonaffected gastric epithelium. By contrast, secretin-containing cells were always present in the normal jejunal mucosa, being located within the epithelium covering villi and the transitional zone. Neurotensin
Metaplastic epithelium did not show neurotensin-containing cells, whereas such cells were observed in two out of the four normal jejunal specimens studied. When present, they were rare (three or four per sample) and were located in the villi. Pancreatic Glucagon and Pancreatic Polpeptide
Cells reacting with antiglucagon and antipancreatic polypeptide (PP) antiserums were found neither in the normal or metaplastic gastric mucosa nor in the normal intestinal epithelium. Discussion
The present data demonstrate that cells containing polypeptide hormones are present in the intestinal metaplasia of the gastric mucosa (Table 1). The polypeptide hormones identified in the metaplastic epithelium included somatostatin, glicentin (gut GLI-I), motilin, and a peptide probably corresponding to cholecystokinin, since it reacted with antigastrin serum before, but not after, absorption with the CCK octapeptide.15 All these hormones are known to be synthesized in the normal human small intestine. With the exception of rare cases,4 the intestinal metaplasia of gastric mucosa does not form villi and resembles the cryptal epithelium of the small intestine.3'5 In this context, it is interesting to note that all endocrine cells demonstrated by immunofluorescence in the metaplastic epithelium are present and tend to concentrate in the normal epithelium of intestinal crypts. 15,17-19 By contrast, endocrine cells which are typically located on '
° Moreover, the greater number of endocrine cells revealed by a nonspecific silver staining, as compared with the cumulative number of immunofluorescent cells, suggests that hormones other than those tested may be present within the metaplastic epithelium.
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Table 1-Occurrence of Different Endocrine Cell Types in Normal Stomach, Intestinal Metaplasia, and Jejunum As Seen in the Present Series of Patients
Normal stomach
Jejunum Hormone
Somatostatin Glicentin Motilin Gastrin Cholecystokinin Secretin Neurotensin Pancreatic glucagon Pancreatic polypeptide *
Oxyntic mucosa Present Absent Absent Absent Absent Absent Absent Absent Absent
Antropyloric Intestinal mucosa metaplasia Present Absent Present Present Absent Absent Absent Absent Absent
Present Present Present Absent* Present Absent Absent Absent Absent
Villi
Crypts
Present Present Present Absent Present Present Present Absent
Presentt Presentt Presentt Absent Presentt Absent Absent Absent Absent
Absent
Uncertain findings in one case (see text).
t More abundant in the crypts than in the villi.
the villi of the normal intestine (ie, cells containing secretin 20 and, except in the dog,2' neurotensin 22), were not detected in the metaplastic epithelium. t This suggests a possible segregation of endocrine cells situated in the epithelium of villi from those located in crypts, and it would be worthwhile to study by immunofluorescence intestinal metaplasia presenting villi to see whether in this case cells containing secretin and neurotensin were present. Addendum Since submission of the manuscript for publication, cells containing cholecystokinin (CCK) have been shown within metaplastic intestinal epithelium of gastric mucosa by use of a specific anti-CCK antiserum.24 References Rubin W, Ross LL, Jeffries GH, Sleisenger MH: Intestinal heterotopia: A fine structural study. Lab Invest 15:1024-1049, 1966 2. Robbins SL: Pathologic basis of disease. Philadelphia, W. B. Saunders Co., 1974 3. Morson BC: Intestinal metaplasia of the gastric mucosa. Brit J Cancer 9:365-376, 1.
1955
Rubin W, Ross LL, Jeffries GH, Sleisenger H: Some physiologic properties of heterotopic intestinal epithelium: Its role in transporting lipid into the gastric mucosa. Lab Invest 16:813-827, 1967 5. Glass GBJ, Pitchumoni CS: Atrophic gastritis: structural and ultrastructural altera4.
fThe absence of secretin-containing cells found in the present investigation by immunofluorescence is at variance with previous ultrastructural studies indicating the occurrence of such cells.12 However, in the latter studies, the identification of secretin-containing cells was based on ultrastructural criteria alone; and it has since become known that other types of small granulated endocrine cells exist in the small intestine, besides those which store secretin.23
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6. 7.
8.
9. 10. 11. 12. 13. 14.
15. 16. 17. 18.
19. 20. 21. 22.
23. 24.
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tions, exfoliative cytology and enzyme cytochemistry and histochemistry, proliferation kinetics, immunological derangements and other causes, and clinical associations and sequellae. Hum Pathol 6:219-250, 1975 Hamperl H: Ober die "gelben (chromaffinen)" Zellen im gesunden und kranken Magendarmschlauch. Virchows Archiv [Pathol Anat] 266:509-548, 1927 Singh I: A note on enterochromaffin cells in islets of ectopic intestinal mucosa in the human. J Anat Soc India 11:57-60, 1962 Polak JM, Coulling I, Doe W, Pearse AGE: The G cells in pernicious anaemia. Gut 12:319-323, 1971 Mitschke H, Becker M: Zur Cytopathologie der disseminierten endokrinen Zellen des Magens bei Gastritis, Ulcus und Magencarcinom. Virchows Archiv [Pathol Anat] 358:81-98, 1973 Rubin W: Endocrine cells in the normal human stomach: A fine structural study. Gastroenterology 63:784-800, 1972 Hage E: Endocrine cells in the metaplastic epithelia, Endocrine Gut and Pancreas. Edited by T Fujita. Amsterdam, Elsevier Scientific Publishing Co., 1976, pp 365-369 Bordi C, Gabrielli M, Missale G: Pathological changes of endocrine cells in chronic atrophic gastritis: An ultrastructural study on peroral gastric biopsy specimens. Arch Pathol Lab Med 102:129-135, 1978 Grimelius LA: A silver nitrate stain for a2 cells in human pancreatic islets. Acta Soc Med Upsal 73:243-270, 1968 Coons AH, Leduc EH, Connolly JM: Studies on antibody production: I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J Exp Med 102:49-60, 1955 Buffa R, Solcia E, Go VLW: Immunohistochemical identification of the cholecystokinin cell in the intestinal mucosa. Gastroenterology 70:528-532, 1976 McGuigan JE: Gastric mucosal intracellular localization of gastrin by immunofluorescence. Gastroenterology 55:315-327, 1968 Pearse AGE, Polak JM, Bloom SR, Adams C, Dryburgh JR, Brown JC: Enterochromaffin cells of the mammalian small intestine as the source of motilin. Virchows Archiv [Cell Pathol] 16:111-120, 1974 Alumets J, Sundler F, Hakanson R: Distribution, ontogeny and ultrastructure of somatostatin immunoreactive cells in the pancreas and gut. Cell Tissue Res 185:465479, 1977 Ravazzola M, Siperstein A, Moody AJ, Sundby F, Jacobsen H, Orci L: Glicentin immunoreactive cells. Their relationship with glucagon producing cells. Endocrinology (Submitted for publication) Larsson LI, Sundler F, Alumets J, Hakanson R, Schaffalitzky de Muckadell OB, Fahrenkrug J: Distribution, ontogeny and ultrastructure of the mammalian secretin cell. Cell Tissue Res 181:361-368, 1977 Orci L, Baetens 0, Rufener C, Brown M, Vale W, Guillemin R: Evidence for immunoreactive neurotensin in dog intestinal mucosa. Life Sci 19:559-562, 1976 Sundler F, Hakanson R, Hammer RA, Alumets J, Carraway B, Leeman SE, Zimmerman EA: Immunohistochemical localization of neurotensin in endocrine cells of the gut. Cell Tissue Res 178:313-321, 1977 Capella C, Solcia E, Frigerio B, Buffa R: Endocrine cells of the human intestine. An ultrastructural study, Endocrine Gut and Pancreas. Edited by T Fujita. Amsterdam, Elsevier Scientific Publishing Co., 1976, pp 43-59 Larsson L-I, Rehfeld C, Stockbrflgger R, Blohme G, Sch88n I-M, Lundqvist G, Kindblom LG, Save-SWderberg J, Grimelius L, Olbe L: Mixed endocrine gastric tumors associated with hypergastrinemia of antral origin. Am J Pathol 93:53-68, 1978
Acknowledgments The authors wish to thank Miss Anne-Marie Lucini, Mrs. Marthe Sidler-Ansermet, and Mrs. Nadine Maalaoui for their skilled technical assistance.
Figure I-Section of a meta-
plastic intestinal epithelium incubated with antisomatostatin antiserum. Several immunofluorescent cells are present at the bottom of a single crypt; the thin apex of the cells reaches the lumen of the crypt. Adjacent crypts appear devoid of immunoreactive cells. (x 140) Figure 2-Section of a metaplastic intestinal epithelium following incubation with antiglicentin antiserum. Several glandular lumens are bordered by numerous fluorescent cells. (x 165) The inset shows the elongated shape of many endocrine cells reaching the glandular
lumen. (x 290) Figure 3Section of intestinal metaplasia treated with antimotilin antiserum. A few imimunoreactive cells are scattered in the epithelium lining glandular profiles. Goblet cells (arrows) are conspicuous. (X 140)
Figure 4A and B-A, section of gastric epithelium after treatment with antigastrin antiserum. The area illustrated contains both normal and metaplastic glands (the latter marked by stars). Normal glands contain more fluorescent cells than those lined by metaplastic epithelium. Two fluorescent cells in the latter are indicated by arrows and shown at higher magnification in the inset. B, section adjacent to that shown in A but incubated with an antigastrin serum adsorbed with cholecystokinin octapeptide. Fluorescent cells are no longer present within metaplastic tissue, while positive cells remain in the unaffected glands. (X180; inset, X230)
Sections of gastric mucosa removed during surgery for cancer or peptic ulcer and containing regions of intestinal metaplasia were studied by the immunofluorescence technique using several antiserums against intestinal hormones. Endocrine cells such as cells containing somatostatin, glicentin (gut GLI-I), motilin, and probably cholecystokinin were found within metaplastic intestinal epithelium while secretin and neurotensin, which are present in the normal intestinal mucosa, were not detected in metaplastic epithelium. The endocrine-cell population present in the intestinal metaplasia resembles that found in the cryptal region of the normal small intestine, a finding in accordance with the fact that intestinal metaplasia of gastric mucosa usually reproduces structural and histochemical characteristics of small intestinal crypts. (Am J Pathol 96:391-398, 1979)
IN CHRONIC INFLAMMATION of the gastric mucosa, the epithelial lining of the gastric glands can be replaced by an epithelium morphologically similar to that normally seen in the small intestine. This phenomenon has been called intestinal heterotopia,' intestinalization,2 or intestinal metaplasia,3 the latter term and its underlying interpretation being now the most widely accepted. Metaplastic intestinal epithelium of the stomach shows a capacity for lipid absorption 4 and an enzymatic content 6 comparable to those of the intestinal mucosa. Histologically, the metaplastic epithelium contains cells such as absorptive, goblet, undifferentiated, and Paneth cells characteristic of the small intestine.1 The occurrence of endocrine cells within metaplastic intestinal tissue has long been recognized.' Using silver staining, previous workers mentioned the argentaffin or enterochromaffin (EC) cell type,7 9 while recent ultrastructural observations 10-12 described additional endocrine cell types on the basis of the specific morphology of secretion granules. However, in view of the difficulty of identifying unambiguously endocrine cell types on ultrastructural criteria alone, we have reinvestigated this problem with immunofluorescence, employing different antiserums against intestinal hormones. This study resulted in the identification of a characteristic pattern of endocrine cells in the metaplastic epithelium. From the Institute of Histology and Embryology, University of Geneva Medical School, Geneva, Switzerland. Supported by Grant No. 3.120.77 from the Swiss National Science Foundation. Accepted for publication March 7, 1979. Address reprint requests to Dr. C. Bordi, Institute of Pathological Anatomy, University of Parma, 1-43100 Parma, Italy.
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Materials and Methods Samples of gastric mucosa were obtained from 13 patients undergoing gastric resection for gastric peptic ulcer (6 cases) or cancer (7 cases). Nine patients were male and 4 female; their age ranged from 41 to 78 years (mean 61.2). In each patient, a strip of gastric mucosa, approximately 2 cm long, was taken from the lesser curvature, from the anterior and from the posterior wall of the oxyntic region, respectively. A fourth strip was taken from the antropyloric region of the lesser curvature, approximately 2 cm above the pylorus. In addition, in 4 patients, a strip of jejunal mucosa was also taken from the transverse section prepared for gastrojejunal anastomosis. The specimens were fixed for 20-24 hours in Bouin's fluid, dehydrated, and embedded in paraffin. Sections of 5 us were stained with hematoxylin-eosin for the identification of metaplastic areas. Successive sections were stained with the silver method of Grimelius 13 to detect most of the endocrine cell types present within the metaplastic epithelium, while other sections were processed for the indirect immunofluorescence technique,"4 with the use of the following antiserums: rabbit antiglicentin (gut GLI-I) (a gift from Dr. A. J. Moody, Copenhagen, Denmark); rabbit antiglucagon 15K (specific for pancreatic glucagon) (a gift from Dr. R. H. Unger, Dallas, Texas); rabbit antibovine pancreatic polypeptide (BPP) (a gift from Dr. R. E. Chance, Indianapolis, Indiana); rabbit antigastrin (a gift from Dr. W. Gepts, Brussels, Belgium); rabbit antisomatostatin and guinea pig antineurotensin (gifts from Dr. S. Ito, Niigata, Japan); rabbit antisecretin and guinea pig antimotilin (gifts from Dr. N. Yanaihara, Shizuoka-Shi, Japan). Most antiserums were used at a 1:50 dilution, except for antimotilin and antisecretin (1: 10), anti-BPP (1 100) and antisomatostatin (1: 200). After incubation with the respective antiserums, the sections were exposed to fluorescein-labeled sheep antirabbit or antiguinea-pig gammaglobulin serums (Pasteur Institute, Paris, France), counterstained with 0.01 % Evans blue, and observed in a Leitz Orthoplan fluorescence microscope equipped with a Ploemopak illuminator. The specificity of the staining reaction was checked in each case by incubating the sections with antiserums previously adsorbed with an excess of their respective antigen. Only immunofluorescence completely abolished by prior incubation of antiserum with the respective antigen was considered in the study. In addition, to discriminate gastrincontaining cells from cholecystokinin (CCK)-containing cells, which appear to stain with most antigastrin serums 15 (including ours), the latter was used before and after adsorption with an excess of CCK octapeptide (Kinevac, kindly donated by Squibb AG, ZUirich, Switzerland).
Results On histologic examination, areas of intestinal metaplasia appeared as surface invaginations lined by an epithelium resembling that of the small intestine. Villous differentiations were not present. Metaplastic areas varied greatly in number and extent from patient to patient and were usually more pronounced in the antropyloric than in the oxyntic region of the
stomach. Endocrine cells were easily identified within metaplastic epithelium by either silver staining or immunofluorescence. Endocrine cells tended to concentrate at the bottom of the invaginations, and they usually showed an elongated shape with a thin apex reaching the tubular lumen (open type). In any sample, the number of endocrine cells varied considerably from one metaplastic region to another even when these were contiguous.
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Also, with any antiserum, areas with fluorescent cells and areas devoid of these cells coexisted in the same patient. The cumulated number of immunofluorescent cells present in a discrete area of the metaplasia was smaller than that of cells stained with Grimelius' silver in the same area, the former usually ranging between 60% and 80% of the latter. Somatostatin
Metaplastic epithelium contained somatostatin-reactive cells in all patients studied, although in highly variable numbers (Figure 1). Moreover, somatostatin-containing cells were always detected in nontransformed areas of the oxyntic and antral mucosa as well as in the normal jejunal epithelium. In antral mucosa these cells were three to four times more numerous than in metaplastic epithelium or in oxyntic and jejunal mucosa. In this latter region fluorescent cells were more frequent in the crypts than in the villi. Glicentin
Positive cells were observed within metaplastic areas of the stomach in all cases except one. Again, the number of glicentin-containing cells was variable, varying from very low (one cell per 3-4 crypts) to very high frequency (numerous cells within the same crypt, as shown in Figure 2). Positive cells were not seen in normal gastric mucosa, but they were seen in jejunal epithelium, especially at the cryptal level. Motilin
Fluorescent cells were found in metaplastic gastric areas (9 out of 13 cases) (Figure 3) and in all jejunal samples studied. In metaplastic epithehum, they were usually fewer than cells containing somatostatin or glicentin. Motilin-containing cells were also found in the uninvolved antropyloric mucosa of 4 patients, but not in oxyntic mucosa. Gastrin-CCK
Cells reacting with antigastrin antiserum were found in metaplastic areas (9 out of 13 cases) as well as in uninvolved antropyloric and in jejunal mucosa (all cases). In the uninvolved antropyloric mucosa these cells were localized in the mid-portion of the glands, and they showed the characteristic round or triangular shape reported for normal antral G cells.'6 On the other hand, fluorescent cells of metaplastic and jejunal epithelium usually appeared more elongated and were located preferentially in the lower part of the crypts (Figure 4A). Adsorption of antigastrin antiserum with the CCK octapeptide did not affect thc immuno-
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fluorescent reaction in cells of the uninvolved antropyloric region but completely abolished that of cells localized in metaplastic or jejunal epithelium (Figure 4B) (except for one case, in which approximately one in every six cells remained fluorescent in the metaplastic epithelium following incubation with the adsorbed antiserum). Secretin
No positive cells were found within metaplastic zones of the stomach, nor in nonaffected gastric epithelium. By contrast, secretin-containing cells were always present in the normal jejunal mucosa, being located within the epithelium covering villi and the transitional zone. Neurotensin
Metaplastic epithelium did not show neurotensin-containing cells, whereas such cells were observed in two out of the four normal jejunal specimens studied. When present, they were rare (three or four per sample) and were located in the villi. Pancreatic Glucagon and Pancreatic Polpeptide
Cells reacting with antiglucagon and antipancreatic polypeptide (PP) antiserums were found neither in the normal or metaplastic gastric mucosa nor in the normal intestinal epithelium. Discussion
The present data demonstrate that cells containing polypeptide hormones are present in the intestinal metaplasia of the gastric mucosa (Table 1). The polypeptide hormones identified in the metaplastic epithelium included somatostatin, glicentin (gut GLI-I), motilin, and a peptide probably corresponding to cholecystokinin, since it reacted with antigastrin serum before, but not after, absorption with the CCK octapeptide.15 All these hormones are known to be synthesized in the normal human small intestine. With the exception of rare cases,4 the intestinal metaplasia of gastric mucosa does not form villi and resembles the cryptal epithelium of the small intestine.3'5 In this context, it is interesting to note that all endocrine cells demonstrated by immunofluorescence in the metaplastic epithelium are present and tend to concentrate in the normal epithelium of intestinal crypts. 15,17-19 By contrast, endocrine cells which are typically located on '
° Moreover, the greater number of endocrine cells revealed by a nonspecific silver staining, as compared with the cumulative number of immunofluorescent cells, suggests that hormones other than those tested may be present within the metaplastic epithelium.
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Table 1-Occurrence of Different Endocrine Cell Types in Normal Stomach, Intestinal Metaplasia, and Jejunum As Seen in the Present Series of Patients
Normal stomach
Jejunum Hormone
Somatostatin Glicentin Motilin Gastrin Cholecystokinin Secretin Neurotensin Pancreatic glucagon Pancreatic polypeptide *
Oxyntic mucosa Present Absent Absent Absent Absent Absent Absent Absent Absent
Antropyloric Intestinal mucosa metaplasia Present Absent Present Present Absent Absent Absent Absent Absent
Present Present Present Absent* Present Absent Absent Absent Absent
Villi
Crypts
Present Present Present Absent Present Present Present Absent
Presentt Presentt Presentt Absent Presentt Absent Absent Absent Absent
Absent
Uncertain findings in one case (see text).
t More abundant in the crypts than in the villi.
the villi of the normal intestine (ie, cells containing secretin 20 and, except in the dog,2' neurotensin 22), were not detected in the metaplastic epithelium. t This suggests a possible segregation of endocrine cells situated in the epithelium of villi from those located in crypts, and it would be worthwhile to study by immunofluorescence intestinal metaplasia presenting villi to see whether in this case cells containing secretin and neurotensin were present. Addendum Since submission of the manuscript for publication, cells containing cholecystokinin (CCK) have been shown within metaplastic intestinal epithelium of gastric mucosa by use of a specific anti-CCK antiserum.24 References Rubin W, Ross LL, Jeffries GH, Sleisenger MH: Intestinal heterotopia: A fine structural study. Lab Invest 15:1024-1049, 1966 2. Robbins SL: Pathologic basis of disease. Philadelphia, W. B. Saunders Co., 1974 3. Morson BC: Intestinal metaplasia of the gastric mucosa. Brit J Cancer 9:365-376, 1.
1955
Rubin W, Ross LL, Jeffries GH, Sleisenger H: Some physiologic properties of heterotopic intestinal epithelium: Its role in transporting lipid into the gastric mucosa. Lab Invest 16:813-827, 1967 5. Glass GBJ, Pitchumoni CS: Atrophic gastritis: structural and ultrastructural altera4.
fThe absence of secretin-containing cells found in the present investigation by immunofluorescence is at variance with previous ultrastructural studies indicating the occurrence of such cells.12 However, in the latter studies, the identification of secretin-containing cells was based on ultrastructural criteria alone; and it has since become known that other types of small granulated endocrine cells exist in the small intestine, besides those which store secretin.23
396
6. 7.
8.
9. 10. 11. 12. 13. 14.
15. 16. 17. 18.
19. 20. 21. 22.
23. 24.
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Acknowledgments The authors wish to thank Miss Anne-Marie Lucini, Mrs. Marthe Sidler-Ansermet, and Mrs. Nadine Maalaoui for their skilled technical assistance.
Figure I-Section of a meta-
plastic intestinal epithelium incubated with antisomatostatin antiserum. Several immunofluorescent cells are present at the bottom of a single crypt; the thin apex of the cells reaches the lumen of the crypt. Adjacent crypts appear devoid of immunoreactive cells. (x 140) Figure 2-Section of a metaplastic intestinal epithelium following incubation with antiglicentin antiserum. Several glandular lumens are bordered by numerous fluorescent cells. (x 165) The inset shows the elongated shape of many endocrine cells reaching the glandular
lumen. (x 290) Figure 3Section of intestinal metaplasia treated with antimotilin antiserum. A few imimunoreactive cells are scattered in the epithelium lining glandular profiles. Goblet cells (arrows) are conspicuous. (X 140)
Figure 4A and B-A, section of gastric epithelium after treatment with antigastrin antiserum. The area illustrated contains both normal and metaplastic glands (the latter marked by stars). Normal glands contain more fluorescent cells than those lined by metaplastic epithelium. Two fluorescent cells in the latter are indicated by arrows and shown at higher magnification in the inset. B, section adjacent to that shown in A but incubated with an antigastrin serum adsorbed with cholecystokinin octapeptide. Fluorescent cells are no longer present within metaplastic tissue, while positive cells remain in the unaffected glands. (X180; inset, X230)
