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Histopathologic and Immunohistochemical Studies on the Mechanism of Interlobular Fibrosis of the Pancreas.

The pathogenesis of chronic pancreatitis is not yet understood. The most common cause of chronic pancreatitis is long-term alcohol abuse.[1] According to our previous study,[2] pancreatic fibrosis associated with alcohol abuse can show any fibrosis pattern according to Martin's 3 main patterns of pancreatic fibroatrophic states[3]: (1) predominantly intralobular sclerosis, which is always homogenous and diffuse; (2) predominantly perilobular sclerosis, with a cirrhosis-like aspect but which is irregular and sometimes patchy; and (3) mixed intralobular and perilobular sclerosis, often homogenously distributed in the gland. Among these 3 patterns, the fibrosis of chronic alcoholic pancreatitis was found mainly in the perilobular or interlobular areas with a nodular lobular appearance.[2]

The most commonly cited theory for the cause of chronic alcoholic pancreatitis suggests the deposition of protein plugs that later calcify, leading to duct obstruction with subsequent fibrotic replacement of the acinar tissue upstream from the occlusion.[4] However, duct obstruction is considered an essential fibrosis mechanism in chronic obstructive pancreatitis distal to a stricture of the pancreatic duct. Chronic obstructive pancreatitis is observed secondary to slow-growing pancreatic carcinomas, ampullary carcinomas, odditis, and pancreatic duct scars[5] and shows a different histologic structure from that of chronic alcoholic pancreatitis. According to our previous study,[6] in chronic alcoholic pancreatitis cases, fibrosis mainly distributed in the interlobular or perilobular areas with a nodular pancreatitis pattern, whereas in pancreatic head carcinomas, which cause complete obstruction of the main pancreatic duct, fibrosis distributed in the interlobular and intralobular areas with lobular atrophy.

In this study, we investigated the mechanism of interlobular fibrosis of the pancreas, which is categorized as chronic alcoholic pancreatitis, based on histologic changes in the pancreatic tissue in patients with ampullary carcinomas with various degrees of stricture of the main pancreatic duct.


Forty patients with ampullary carcinomas (mean age, 63 years), treated with surgery at Juntendo University Hospital, Tokyo, Japan, during the past 10 years, were studied histopathologically. None of the patients included in this group drank heavily. For each patient with ampullary carcinoma, 2 to 7 blocks were taken from the pancreatic tissues, fixed in a formaldehyde solution, dehydrated, and embedded in paraffin. Histologic sections were stained with hematoxylin-eosin, Masson's trichrome, and elastica van Gieson and were immunostained for anti-[Alpha]-smooth muscle actin ([Alpha]-SMA; clone 1 A 4, Dako, Tokyo, Japan) as a marker for myofibroblasts,[7,8] MIB1 (Immunoteck SA, Marseille, France) cell cycle detection, and anti-pancreatic polypeptide, which is selectively distributed in the ventral pancreas.[9,10] The TUNEL method was used for apoptotic cells.

Quantitative analysis was performed using an image analyzer (NIH image version 1.55) after taking photographs. Both MIB1-positive and apoptotic cell ratios were determined per 1000 acinar cells counted. The data are represented as mean [+ or -] SD. The significance of differences in both MIB1-positive and apoptotic cell ratios was tested using the Mann-Whitney U test. P values of less than .05 were regarded as statistically significant.

Histologic findings were evaluated in a pancreatic polypeptide-rich area, named the ventral pancreas, because ampullary carcinomas are anatomically involved in the outflow of the pancreatic juice from Wirsung's duct or the main pancreatic duct but not from Santorini's duct or the accessory pancreatic duct. Histologic changes in fibrosis were divided into 3 categories as follows: mild change showing rather diffuse distribution of mild interlobular fibrosis, seldom accompanied by acinar atrophy; moderate change revealing moderate interlobular up to moderate intralobular fibrosis with acinar atrophy; and marked change showing marked interlobular up to marked intralobular fibrosis with acinar atrophy.

In addition, 20 patients with clinically diagnosed chronic alcoholic pancreatitis (mean age, 52 years) at Juntendo University Hospital were included in this study. Tissue specimens were obtained during surgical resection. Each of the 20 patients with chronic alcoholic pancreatitis had consumed an alcohol amount equal to more than 100 g of 100% ethanol daily for more than 20 years and was considered a heavy drinker.


Fibrosis with and without acinar atrophy was observed in 23 of 40 patients with ampullary carcinomas and was classified into 3 categories: mild changes (10), moderate changes (9), and marked changes (4), as summarized in Table 1. In most cases, these changes were found in the ventral pancreas but not in the dorsal pancreas. In these 23 patients, the main pancreatic duct was considered to be strictured or obliterated to various degrees due to the ampullary carcinoma.

Table 1. Histologic Changes and Quantitative Analysis of the Pancreatic Tissues in Patients With Ampullary Carcinomas
 No. of
Histologic Changes Cases(*) Fibrosis Distribution

Almost normal 17 (11) None
Mild 10 (8) Interlobular
Moderate 9 (4) Interlobular and intralobular
Marked 4 (3) Interlobular and intralobular

 MIB1 Ratio
Histologic Changes Acinar Atrophy (Acini), [salinity]

Almost normal None 2.0 [+ or -] 1.9
Mild None/mild 4.1 [+ or -] 3.2
Moderate Mild/moderate 2.5 [+ or -] 1.5
Marked Moderate/marked 2.2 [+ or -] 2.2

 Apoptosis Ratio
Histologic Changes (Acini), [salinity]

Almost normal 0.3 [+ or -] 0.5
Mild 0.5 [+ or -] 1.3
Moderate 0.9 [+ or -] 1.0
Marked 2.0 [+ or -] 1.0([dagger])

(*) Data in parentheses represent quantitative analysis cases for MIB1-positive and apoptotic cell ratios.

([dagger]) Significant difference with almost normal cases (P < .05).

In cases with mild changes, fibrosis was distributed diffusely to a mild degree in the interlobular area (Figure 1). The lobules seldom showed atrophy and seemed to be rather expansive. In cases with moderate changes, fibrosis was moderate in both the interlobular and intralobular areas with mild acinar atrophy, resulting in atrophy of the lobules. In cases with marked changes, fibrosis was found in the interlobular area and loosely in the intralobular area, acinar atrophy was also prominent (Figure 2), and the main pancreatic duct was dilated.


By quantitative analysis, the mild change cases showed both a higher MIB1-positive cell ratio (4.1 [salinity] [+ or -] 3.2 [salinity]) and a lower apoptotic cell ratio (0.5 [salinity] [+ or -] 1.3 [salinity]) among the acinar cells than those of the moderate and marked change cases, without statistical significance (Table 1).

In all cases of chronic alcoholic pancreatitis, fibrosis was patchily distributed in the interlobular or perilobular areas with the "nodular pancreatitis" pattern[3] (Figure 3), and in some advanced cases, in which protein plugs and pancreatic stones were also found, fibrosis extended into the intralobular areas (Figure 4).


The immunoreactivity of anti-[Alpha]-SMA in mild change patients with ampullary carcinomas was lightly distributed in periductal fibrosis and was found very scant in interlobular fibrosis without continuity between the periductal and interlobular areas. On the contrary, in cases with moderate or advanced changes, marked immunoreactivity was observed in the interlobular area and in the area surrounding both the acini and intralobular ducts (Table 2). No anti-[Alpha]-SMA immunoreactivity, except in the periductal tissue, was found in cases without fibrosis. Anti-[Alpha]-SMA immunoreactivity was found in interlobular fibrosis in patients with chronic alcoholic pancreatitis.

Table 2. Distribution of Anti-[Alpha]-Smooth Muscle Actin ([Alpha]-SMA) Immunoreactivity in Patients With Ampullary Carcinomas
 Anti-[Alpha]-SMA-Positive Area in Fibrosis(*)

Histologic Change Periductal Interlobular Intralobular

Almost normal + - -
Mild + [+ or -] -
Moderate + + +
Marked ++ ++ ++

(*) Minus sign indicates negative; [+ or -], very scant; +, positive; and ++, markedly positive.


In the present study, the pancreatic tissues in patients with ampullary carcinomas showed various changes, ranging from almost normal to typical patterns of chronic obstructive pancreatitis.[5] Some patients simply had interlobular fibrosis with a rather expansive lobular appearance, whereas other patients showed marked interlobular and intralobular fibrosis with atrophy of the lobules. The former may be caused by incomplete obstruction of the main pancreatic duct, whereas the latter occurred as a result of almost complete obstruction of the main pancreatic duct, similar to that in patients with carcinoma of the head of the pancreas.[6] In most patients, these changes were found only in the ventral pancreas, in which the pancreatic juice flows to the major duodenal papilla, and not in the dorsal pancreas in the head portion,[9,10] which drains from the minor papilla via the accessory pancreatic duct. Hence, these changes resulted from a disturbance in the outflow of the pancreatic juice.

In this study, cell kinetics and apoptotic cell counts revealed that the acinar cells of the mild change cases had a higher MIB1-positive ratio and lower apoptotic cell ratio than those of both the moderate and marked change cases. It is known that experimental pancreatic duct ligation causes acinar cell deletion, distal pancreatic atrophy, and duct proliferation in mammals.[11,12] The acinar cell deletion in such models is the result of cell death, morphologically termed apoptosis. Hence, these pathologic changes closely resembled or were equal to those cases with both moderate and marked changes. It is also known that acinar regeneration is extremely limited. Thereby, the higher MIB1-positive ratio of the acini together with the lower apoptotic ratio in this study may be due to a certain compensation of the

acinar cells against incomplete duct obstruction. Nevertheless, these findings were reasonable findings with regard to the expansive appearance of the lobuli, which may finally lead to a nodular lobular pattern.

Immunoreactivity of anti-[Alpha]-SMA, which is a marker for myofibroblasts,[7,8] was independently found both in the periductal space and at very low levels in interlobular fibrosis in cases with mild changes. It is common knowledge that myofibroblasts play an important role in not only wound healing but also collagenization, which is followed by fibrosis in many organs.[13-15] Recently, the identification of pancreatic stellate cells was reported by Bachem et al,[16,17] and these cells, also called vitamin A-storing cells, when isolated from the pancreas were shown to differentiate into myofibroblast-like cells that express [Alpha]-SMA and produce type I and III collagen, laminin, and fibronectin. Hence, in the mild change cases, the scant anti-[Alpha]-SMA immunoreactivity in the interlobular areas, but not in the pancreatic duct, was considered the beginning of interlobular fibrosis.

In this study, fibrosis in chronic alcoholic pancreatitis was patchily distributed in the interlobular or perilobular areas with anti-[Alpha]-SMA immunoreactivity and nodular lobular patterns. Hence, the cases with mild changes in patients with ampullary carcinomas that showed diffuse mild interlobular fibrosis with scant anti-[Alpha]-SMA immunoreactivity and expansive lobular patterns had histologic findings similar to those of patients with chronic alcoholic pancreatitis, except for excessive fibrosis with patchy distribution. Such different fibrosis distribution can be clearly explained as follows: in ampullary carcinoma cases, duct obstruction was found at the terminal portion of the main pancreatic duct, whereas in chronic alcoholic pancreatitis cases, protein plugs may "plug" the small ductules, leading to obstruction of the area drained by these ductules, thus inducing a nodular pancreatitis pattern. As for excessive fibrosis, the period of alcohol intake necessary for the development of chronic alcoholic pancreatitis has been estimated to be more than 10 years. Moreover, some patients with ampullary carcinomas had accompanying pancreatolithiasis.[18-20] The pathogenesis of this condition is explained by tumor stenosis of the main pancreatic duct, stagnation of the pancreatic juice, and constitutional changes in the pancreatic juice. Hence, the pancreatic histologic findings due to duct obstruction were mimicking that of chronic alcoholic pancreatitis.

In conclusion, incomplete obstruction of the main pancreatic duct caused diffuse, mild interlobular fibrosis, which was accompanied by both anti-[Alpha]-SMA immunoreactivity and an expansive lobular appearance. These findings were similar to those of chronic alcoholic pancreatitis cases. Moreover, Tanaka et al[21] demonstrated experimentally histologic findings similar to those of human chronic alcoholic pancreatitis by combining alcohol administration with incomplete ductal obstruction. Therefore, such incomplete obstruction of the pancreatic duct plays an important role in the beginning of interlobular fibrosis, which is categorized as chronic alcoholic pancreatitis.

This work was supported by a research grand from the Ministry of Health and Welfare, Japan. We thank Hiroshi Abe for skillful technical assistance and Kaori Kagoshima for typing the manuscript.


[1.] Singh M, Sinsek H. Ethanol and the pancreas: current status. Gastroenterology. 1990;98:1051-1062.

[2.] Suda K, Shiotsu H, Nakamura T, Akai J, Nakamura T. Pancreatic fibrosis in patients with chronic alcoholic abuse: correlation with alcoholic pancreatitis. Am J Gastroenterol. 1994;89:2060-2062.

[3.] Martin ED. Different pathomorphological aspects of pancreatic fibrosis, correlated with etiology: anatomical study of 300 cases. In: Gyr KE, Singer MV, Sarles H, eds. Pancreatitis: Concepts and Classification. Amsterdam, the Netherlands: Elsevier; 1984:77-82.

[4.] Sarles H. Chronic pancreatitis: etiology and pathophysiology. In: Go VLW, Gardner JD, Brooks EP, Lebenthal E, Dimagno EP, Scheele GA, eds. The Exocrine Pancreas: Biology, Pathobiology, and Disease. New York, NY: Raven Press; 1986: 525-575.

[5.] Sarhel J, Sarles H. Chronic calcifying and obstructive pancreatitis: two entities. In: Gyr KE, Singer MV, Sarles H, eds. Pancreatitis: Concepts and Classification. Amsterdam, the Netherlands: Elsevier; 1984:47-49.

[6.] Suda K, Mogaki M, Oyama T, Matsumoto Y. Histopathologic and immunohistochemical studies on alcoholic pancreatitis and chronic obstructive pancreatitis: special emphasis on ductal obstruction and genesis of pancreatitis. Am J Gastroenterol. 1990;85:271-276.

[7.] Darby I, Skalli O, Gabbiani G. [Alpha]-smooth muscle is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest. 1990;63:21-29.

[8.] Zhang K, Rekhter MD, Gordon D, Phan SH. Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis: a combined immunohistochemical and in situ hybridization. Am J Pathol. 1994;145:114-125.

[9.] Kloppel G, Lenzen S. Anatomy and physiology of the endocrine pancreas. In: Kloppel G. Heitz PU, eds. Pancreatic Pathology. London, England: Churchill Livingstone; 1984:133-153.

[10.] Suda K, Mizuguchi K, Hoshino A. Differences of the ventral and dorsal anlagen of pancreas after fusion. Acta Pathol Jpn. 1981;31:583-589.

[11.] Walker NI. Ultrastructure of the rat pancreas after experimental duct ligation, I: the role of apoptosis and intraepithelial macrophages in acinar cell deletion. Am J Pathol. 1987;126:439-451.

[12.] Walker NI, Winterford CM, Kerr JFR. Ultrastructore of the rat pancreas after experimental duct ligation, II: duct and stromal cell proliferation, differentiation, and deletion. Pancreas. 1992;7:420-433.

[13.] Bhathal PS. Presence of modified fibroblasts in cirrhotic livers in man. Pathology. 1972;4:139-144.

[14.] Nakano M, Worrier TM, Lieber CS. Perivenular fibrosis in alcoholic liver injury: ultrastructive and histologic progression. Gastroenterology. 1982;83:777-785.

[15.] Eyden BP, Ponting J, Davies H, Bartley C, Torgersen E. Defining the myofibroblast: normal tissues, with special reference to the stromal cells of Wharton's jelly in human umbilical cord. J Submicrosc Cytol Pathol. 1994;26:347-355.

[16.] Bachem MG, Sell KM, Melchior R, Kropf J. Tumor necrosis facter alpha (TNF-[Alpha]) and transforming growth factor [Beta] 1 (TGF-[Beta] 1) stimulate transdifferentiation of fat-storing cells into myofibroblasts and fibronectin synthesis. Virchow Arch B Cell Pathol. 1993;63:123-130.

[17.] Bachem MG, Schneider E, Gro[Beta] H, et al. Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology. 1998;115:421-432.

[18.] Oguchi H, Nagata A, Hirabayashi H, et al. A study on the complication of malignancy in pancreatic lithiasis. Jpn J GastroenteroL 1982;79:851-857.

[19.] Ogiso S, Nimura Y, Okamoto K, et al. Carcinoma of the papilla of Vater associated with pancreatolithiasis: a case report. Jpn J Gastroenterol. 1989;86: 2456-2459.

[20.] Ochiai M, Sato T, Fujino MA, et al. A case of carcinoma of the duodenal papilla associated with pancreatolithiasis. J Jpn Pancreas Soc. 1994;9:387-390.

[21.] Tanaka T, Miura Y, Matsugu Y, Ichiba Y, Ito H, Dohi K. Pancreatic duct obstruction is an aggravating factor in the canine model of chronic alcoholic pancreatitis. Gastroenterology. 1998;115:1248-1253.

Accepted for publication March 9, 2000.

From the Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan.

Reprints: Koichi Suda, MD, Department of Pathology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan (e-mail:
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Author:Suda, Koichi; Takase, Masaru; Takei, Kazuo; Kumasaka, Toshio; Suzuki, Fujihiko
Publication:Archives of Pathology & Laboratory Medicine
Geographic Code:1USA
Date:Sep 1, 2000
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