Exocrine Pancreatic Insufficiency Syndrome in CBA/J Mice I. Ultrastructural Study John J. Eppig, PhD, and Edward H. Leiter, PhD

The pathogenesis of a spontaneously occurring exocrine pancreatic insufficiency (EPI) syndrome in CBA/J mice was studied at the ultrastructural level. Initial cytologic manifestations of this syndrome are seen as a progressive digestion of the zymogen granules, beginning at the periphery and proceeding toward the granule interior. Granule membrane breakdown, fusion of neighboring granules, and a release of zymogen contents into the cytoplasm are frequently observed in later stages; in some cases the entire granule contents appear digested before membrane breakdown is observed. In either case, pathologic changes are subsequently observed in mitochondria and rough endoplasmic reticulum. Remnants of lysed cells are then engulfed by invading macrophages, and infiltration by fat cells is observed. Secretory ducts and islets of Langerhans show no pathologic changes even after total autolysis of the exocrine pancreas. Morphologic evidence showing zymogen granule destabilization, coupled with biochemical evidence presented in an accompanying paper, indicate that intracellular autodigestion is the mechanism of exocrine cell death. (Am J Pathol 86:17-30. 1977)

VARYING DEGREES OF NECROSIS of the exocrine pancreas in laboratorv mice and rats can be induced by several different experimental procedures, specifically DL-ethionine feeding,' DL-ethionine feeding in combination with protein-deficient 2 and choline-deficient3 diets, and prolonged fasting.' Rapid and massive exocrine cell necrosis and atrophy can also be experimentally induced in mice by introduction of a wide variety of viruses, notably Group B Coxsackie virus,5 Venezuelan equine encephalomyelitis virus,6 reovirus,7 strain E encephalomyocarditis (EMC) virus,8 and infectious pancreatic necrosis virus.' However, until the report of Pivetta and Green 10 of a spontaneously occurring pancreatitis in CBA/J mice, a model for studying genetic predisposition to pancreatitis in the mouse did not exist. Screening 15,462 culls of 13 different inbred strains in the Production Colonies of The Jackson Laboratory, these workers found three CBA/J mice with clinical From The Jackson Laboraton, Bar Harbor, Maine. Supported by Grants ANM-17631 from the National Institute of Arthritis, NMetabolism, and Digestive Diseases and HD-(5944 from the National Institute of Child Health and Human Development. The Jackson Laboraton is fullv accredited bv the American Association for Accreditation of Laboratorv Animal Care. Accepted for publication August 17, 1976. Address reprint requests to Dr. Edward H. Leiter, The Jackson Laboratonr, Bar Harbor. ME

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symptoms of exocrine pancreatic insufficiency (runting, fatty yellow feces with almost no trypsin-like activity, massive degeneration of the acinar pancreas with replacement by fat, and normal pancreatic islets of Langerhans). The spontaneous exocrine pancreatic disease in CBA/J mice closely resembled the "juvenile atrophy" of the canine pancreas 11,12 with regard to a) the lack of both fibrous tissue and severe inflammatory involvement at the same time that massive nonhemorrhagic necrosis of the exocrine pancreas is occurring; b) the retention of islets of Langerhans, major ducts, vessels, and nerves; and c) predisposition of the disease for one breed (German Shepherd) or strain (CBA/J). As in the case of canine juvenile pancreatic atrophy, the symptoms occurring in CBA/J mice can be ameliorated by pancreatin supplementation. Unlike these two animal models of pancreatic disease, in the cases reported by Kendrey and Row 13 of a spontaneous chronic pancreatitis in three inbred strains of rats, there was heavy fibrosis of the connective tissue, dilation of the ducts, and vascular lesions similar to polyarteritis nodosa in man. Preliminary breeding tests with the siblings of the affected mice, performed by Pivetta and Green,10 led them to infer that the disease was produced by an autosomal recessive mutation which they designated exocrine pancreatic insufficiency (epi). Their genetic analysis was based upon detection of the trait in juveniles of the CBA/J strain between 2 to 3 weeks of age; these workers were, in effect, studying acute juvenile atrophy of the pancreas. Consequently, their analysis was complicated by diagnostic difficulties, since both runting due to maternal milk deficiencies and yellow feces are not uncommon events in unselected litters of young CBA/J mice. In addition to the problem of distinguishing between runting due to pancreatic disease versus inadequate maternal nourishment in CBA/J juveniles, cases of juvenile pancreatic necrosis were acute in nature, with massive exocrine cell necrosis having occurred by the time clinical symptoms were diagnostic. Thus, little information regarding the primary ultrastructural and biochemical events underlying acinar cell autolysis could be obtained from these severely diseased juveniles, most of which died within days after being diagnosed. To characterize the disease progression at the ultrastructural level, we have taken advantage of the continued appearance of this disease at a low frequency in 1- to 4-month old CBA/J mice in the Production Colony at the Jackson Laboratory. In contrast to the use of moribund preweaning affected mice, the use of diseased postweaning mice culled from the Production Unit breeding population is advantageous for analysis because these mice exhibit a less precipitous decline in health (probably due to the larger mass of the acinar pancreas at the time of disease onset) and thus

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present a full spectrum of the stages of exocrine pancreatic atrophy for ultrastructural analysis and not simply the terminal stage. An additional advantage of analyzing the disease in adults is that confidence in the diagnosis can be achieved by supplementing the animal's diet with pancreatic enzymes, with resultant restoration of the normal body weight, healthy coat condition, and fertility. This enzyme therapy confirms exocrine pancreatic insufficiency as the diagnosis. While enzyme therapy has enabled us to achieve successful matings of affected with affected (presumed homozygote epi/epi X epi/epi), the data from these matings have not supported the concept of trait transmission as a simple autosomal recessive mutation with complete penetrance. Therefore, we shall refer to exocrine pancreatic insufficiency disease in CBA/J mice as the EPI syndrome rather than using the genetic designation (epi/epi) proposed by Pivetta and Green.10 However, there is clearly an involvement of genetic background in the etiology of the syndrome, since it occurs spontaneously only in the CBA/J inbred strain and all of our attempts to transmit this disease to other inbred strains of mice by genetic outcrossing or by other means have been unsuccessful. Materials and Methods CBA/J mice ranging in age from 3 weeks to 4 months were selected from animals culled from the stocks of the Jackson Laboratorv Production Department. The mice had been culled on the basis of the appearance of diagnostic symptoms such as weight loss, runting, and fattv vellow feces. Some of these mice were sacrificed and samples fixed for analysis shortly after being culled from the stock population, while others were maintained for i to 4 weeks on diet 96 W (Emorv-Morse, Co., Old Guilford, Conn.) to which 1.75% (w, w) pancreatin (Parke-Davis Pateric granules) was added. The pancreatin treatments do not arrest cytolysis or the progress of the svndrome but do alleviate the symptoms. Most pancreatin-maintained mice were found to have no exocrine pancreas. Control pancreases were taken from a subline of CBA/J mice maintained as a separate, isolated colonv by Dr. Edwin Les of the Jackson Laboratory. This isolated colony has been free of diagnostic symptoms of the disease. The pancreases were diced into pieces 0.25 to 0.50 mm on a side while immersed in icecold 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3). The pieces were transferred to fresh glutaraldehyde solution where they were fixed for 2 to 3 hours at 4 C. Samples were then rinsed overnight in cold 0.1 M sodium phosphate buffer, pH 7.3, containing 7%c sucrose, and postfixed in 19c osmium tetroxide in the same buffer-sucrose solution at 4 C. The samples were dehydrated in an ethanol series and propylene oxide and embedded in Epon. Sections with gray to silver diffraction colors were stained with uran-l acetate and lead citrate and examined with a Hitachi HU-IIC electron microscope operated at 75 kW.

Results The overall structure of a pancreatic exocrine cell from an undiseased CBA/J mouse is shown in Figure 1. One or two nuclei are found in the basal region of the cell, and mitochondria are distributed throughout the

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cytoplasm. There is an abundance of rough endoplasmic reticulum (RER), often dilated with flocculent material, that is seen both in cisternal and in tubular form. Golgi complexes are located in the apical region of the cell, and condensing vacuoles are frequently associated with them. The zymogen granules, which are about 1.1 to 1.2 ,u in diameter, have a homogeneous electron density. The earliest indication of cytopathology we have observed is a decrease in the electron opacity of some zymogen granules, suggesting degradation proceeding from the periphery toward the center of the granules. The number of granules showing this condition varies from cell to cell in affected animals. Adjacent cells often show variable progress of the granule digestion (Figure 2). During the early stages of granule digestion, the granule's limiting membrane appears to remain intact. In some cases the zymogen contents of a granule appear completely digested without visible effects on the granule membrane. Generally, however, the membrane does show some degree of lytic change, and when sufficient lysis occurs membrane fusion between neighboring zymogen granules is observed (Figure 3). At this stage of severe membrane destabilization, zymogen granule contents are often seen spread across the ruptured granule membrane and out into the cytoplasm (Figure 4A). Whorled lamellar membranous structures are sometimes found within the zymogen material, suggesting lysis of other cytoplasmic structures (Figure 4B). We have not observed any decrease in the number of mature zymogen granules or in condensing vacuoles in the pancreatic acinar cells of diseased mice. During the initial stages of granule digestion, the RER appears normal (Figures 2 and 3). However, as digestion of the granules proceeds, the RER becomes fragmented and vesiculated (Figure 5). Further, mitochondria become swollen and degenerate (Figure 5). Sometimes, the RER cisternae are seen whorled around zymogen granules. Eventually digestive processes result in extensive cytolysis. In regions of the pancreas where this has occurred, there is an invasion of phagocytic cells which engulf and digest the remains of the exocrine cells (Figure 6). Subsequently, there is a substitution with fatty tissue which surrounds the remaining pancreatic ducts and endocrine islets of Langerhans. The ducts and endocrine cells of the pancreas appear unaffected by the total acinar cell cytolysis which occurs around them. The fatty tissue- appears without concomitant fibrosis or calcification of the pancreas. Discussion

Our observation of degenerative changes in the zymogen granules prior to extensive disorganization of the rough endoplasmic reticulum and

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other formed elements in the acinar cell cytoplasm clearly differentiates the ultrastructural pathogenesis of the exocrine pancreatic insufficiencv disease in CBA/J mice from acinar cell atrophy induced by ethionine intoxication ` or food restriction.' In acinar cell atrophy experimentally induced in C57BL/10J mice during fasting, Nevalainen and Janigan 4 observed a decrease in both the size and number of zymogen granules within 24 hours of fasting, and after 3 days, focal cytoplasmic degeneration, particularly at the level of the rough endoplasmic reticulpm. Similarlv, in rats fed a protein-deficient diet supplemented with DL-ethionine, Herman and Fitzgerald 2 observed the earliest degenerative changes to occur in the endoplasmic reticulum. Signs of intracellular destabilization of zymogen granules were not reported in either of these studies, and unlike the massive necrosis of exocrine pancreas encountered in the EPI syndrome, only focal necrosis was observed in both of the above studies. We conclude from the unique ultrastructural features of the EPI svndrome that the apparent autodigestion of zymogen granules is an earlv step in the mechanism which results in cell lysis. In the accompanying paper,14 we provide biochemic4l data showing premature activation of trypsinogen and chymotrypsinogen in the isolated zymogen granule fraction but not in the RER of diseased mice. This demonstration, coupled with our inability to detect histologically any evidence for reflux of pancreatic juice or bile back into the pancreas as a result of pancreatic or bile duct obstruction, confirms intracellular autodigestion initiated at the zymogen granules as the mechanism of exocrine cell death. A similar massive autodigestive pathology in mice has recently been described at the ultrastructural level by Lombardi et al.,3 who induced massive hemorrhagic pancreatitis in 1-month-old female Swiss Webster mice with 0.5% DL-ethionine in combination with a choline-deficient diet. Interestingly, while the sequence of ultrastructural alterations reported by these workers differed from those seen in this paper (i.e., alterations in the normal structure of the RER prior to zymogen granule degeneration), the later stages of this experimental pancreatitis were characterized by the same degradation of zymogen granule contents and limiting membranes as reported in this study. Lombardi et a.3 ascribed the experimental pancreatitis to an intraparenchymal activation of zymogens. While ethionine is generally hypothesized to effect pancreatic lesions through effects on RNA and protein metabolism, these workers3 point out that a primary involvement of a derangement in phospholipid metabolism is implicated under their experimental conditions of choline deficiency. We have no data bearing on the question of phospholipid metabolism in normal versus diseased exocrine pancreatic cells, but we cannot discount the possibility

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that a defect in the zymogen granule membrane is an initiator of the syndrome. Although the activation of pancreatic zymogens in the intestine is normally mediated by trypsin, it is not known whether trypsin is involved in the intrapancreatic activation of zymogens in CBA/J mice. High levels of active trypsin were found in the pancreas of diseased mice.14 Nevertheless, we observed that not all zymogen granules in diseased cells undergo autodigestion. Assuming that there is not a compartmentalization of zymogens in different zymogen granule populations, it may be expected that normal trypsinogen is synthesized in these cells and that the high levels of trypsin detected biochemically are primarily due to activated trypsinogen. The premature intracellular activation of a small amount of trypsinogen would autocatalytically activate more trypsinogen and, in turn, activate other zymogens such as prophospholipase A. This defective premature activation of zymogens may represent a mutation in either structural or regulatory genes for an intrapancreatic trypsin inhibitor (see review by Trautschold et al.15) or a defect in the secretory granule membrane. An effect of genetic background on susceptibility to this disease is suggested by the fact that, of 13 inbred strains of mice screened for the disease, only the CBA/J strain exhibits it spontaneously and at a reasonable frequency. However, it seems unlikely that the disease etiology is a simple genetic mutation since we cannot maintain the disease from generation to generation by planned breeding programs. While we cannot strictly rule out an autoimmune reaction as the cause for the selective destruction of the exocrine pancreas, the ultrastructural changes in the zymogen granules followed by intracellular digestion can be observed well before macrophages and other cells involved in the inflammatory response invade the tissue. Light microscopic analysis corroborated that acinar cell autolysis preceded the inflammatory response. An alternative cause of the disease could be viral infection; the remarkable resemblance between the disease and pancreatic pathologies in mice produced by experimental Coxsackie B5 virus infection has been described by Lansdown.16 While we have been unable to detect Coxsackie virus in CBA/J mice, we have indications of oncornavirus activity in the exocrine pancreas of this strain. High titers of Type C group-specific viral antigen (GS), as detected by complement fixation test, have been detected in sonicated pancreases and other tissues of CBA/J mice which have produced diseased mice (therefore, presumed heterozygotes if the disease were transmitted as a Mendelian gene). Paradoxically, electron microscopic examination does not reveal mature C particles but, instead, occa-

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sional intercisternal Type A particles (Figure 3B), almost always in exocrine cells. While we have no evidence as yet that might link the presence of oncornavirus to the destruction of pancreatic exocrine cells, it is of interest to note that Gardner et al."7 have demonstrated by experimental transmission an indigenous Type C virus to be the agent responsible for degeneration of spinal cord neurons in a spontaneous lower limb paralysis disease in mice. This demonstration of a cytopathologic effect of murine oncornavirus, coupled with a recently described genetic control of Type C virus production in certain inbred mice strains,"8 makes it advisable to pursue further the question of a potential role of virus in the EPI syndrome. In summary: While the precise genetic and/or extrachromosomal factors involved in the etiology of the EPI syndrome are as yet unresolved, this spontaneously occurring disease in CBA/J mice provides an excellent model for studying the genetic and environmental factors that contribute toward a defective secretorv control mechanism in exocrine pancreatic insufficiencv. References 1. Wachstein M, Meisel E: Equal effectiveness of L- and D-ethionine in producing tissue damage in rats and mice. Proc Soc Exp Biol Med 82:70-72, 1953 2. Herman L, Fitzgerald PJ: The degenerative changes in pancreatic acinar cells caused by dl-ethionine. J Cell Biol 12:277-296, 1962 3. Lombardi B, Estes LW, Longnecker DS: Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by DL-ethionine fed with a cholinedeficient diet. Am J Pathol 79:465-476, 1975 4. Nevalainen TJ, Janigan DT: Degeneration of mouse pancreatic acinar cells during fasting. Virchows Arch [Zellpathol] 15:107-117, 1974 5. Pappenheimer ANM, Kunz LJ, Richardson S: Passage of Coxsackie virus (Connecticut-5 strain) in adult mice with production of pancreatic disease. J Exp Med 94:45-64, 1951 6. Gorelkin L Jahrling PB: Pancreatic involvement by Venezuelan equine encephalomvelitis virus in the hamster. Am J Pathol 73:349-356, 1974 7. W'alters NI-NI, Joske RA, Leak PJ, Stanley NF: Murine infection with reovirus. I. Pathology of the acute phase. Br J Exp Pathol 44:427-436, 1963 8. Craighead JE: Pathogenicity of the NI and E variants of the encephalomrocarditis (ENMC) virus. II. Lesions of the pancreas, parotid, and lacrimal glands. Am J Pathol 48:375-386, 1966 9. Angiolilli RF, Rio GJ: Infectious pancreatic necrosis virus-induced pancreatic lesions in Swiss albino mice: Electron microscopy. Am J Vet Res 34:1513-1520, 1972 11.

Pivetta OH, Green EL: Exocrine pancreatic insufficiency: A new recessive mutation in mice. J Hered 64:301-302, 1973 Anderson N, Low D: Juvenile atrophy of the canine pancreas. Anim Hosp

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1:101-109, 1965 Anderson N, Low D: Diseases of the canine pancreas: A comparative studv of 103

10.

cases. Anim Hosp 1:189-194, 1965

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13. Kendrey G, Roe FJC: Histopathological changes in the pancreas of laboratory rats. Lab Anim 3:207-220, 1969 14. Leiter E, Dempsey E, Eppig J: Exocrine pancreatic insufficiency syndrome in CBA/J mice. II. Biochemical studies. Am J Pathol 86:3146, 1977 15. Trautschold I, Werle E, Zickgraf-Rfldel G: Concerning the kallikrein-trypsin inhibitor. Arzneim Forsch 16:1507-1515, 1966 16. Lansdown ABG: Exocrine pancreatic insufficiency: A comparison of the clinical findings with the epi/epi mutation and Coxsackie virus B infection in mice. J Hered 65:378, 1974 17. Gardner MB, Henderson BE, Officer EJ, Rongey RW, Parker JC, Oliver C, Estes JD, Huebner RJ: A spontaneous lower motor neuron disease apparently caused by indigenous type-C RNA virus in wild mice. J Natl Cancer Inst 51:1243-1254, 1973 18. Boiocchi M, Della Torre G, Della Porta G: Genetic control of endogenous C-type virus production in pancreatic acinar cells of C57BL/He and C57BL/6J mice. Proc Natl Acad Sci USA 72:1892-1894, 1975

Acknowledgments We wish to express our deepest gratitude to those animal caretakers in the Morrell Park Production Unit of the Jackson Laboratory who screened large numbers of CBA/J mice to provide us with the diseased animals used in this study. We thank Dr. Eva Eicher of this Laboratory for the information that diseased animals could be maintained on a pancreatin-supplemented diet and Lester Bunker for his excellent technical assistance. Finally, we are grateful to Dr. Edwin Les of this Laboratory for generously providing control mice.

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Fur 1-Normal pancreatic acinar cells from CBA/J mouse. These cells are usually characterized by abundant rough endoplasmic reticulum (RER), scattered mitochondria (M), and Golgi complexes (G) with collcting vacuoles which are present in the apicial region. The nucleus (N) is basaily located. This particular cell is binucleate. An occasional lysosome (L) is usually seen in the perinuclear region. The zymogen granules (Z) are of uniform electron density. (x 8250)

Figure 2-Initial cytopathology observed in CBA/J pancreatic acinar cells is a digestion of the zymogen granule progressing from the periphery to the interior (arrows). RER and mitochondria (M) do not appear affected at this stage. A lower percentage of the granules are affected in Cell A than in Cell B. (x 12,200)

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Fge 3J-As zymogen granub digestion proceeds, limiting membranes become disrupted, some granules fuse, and digestive material leaks into the cytoplasm. At the single arrow, the granule membrane has become discontinuous, though not completely digested, and zymogen granule contents have leaked out. At the double arrow the contents of a granule appear to be flowing into a vacuole which may be the remainder of a more completely digested granule. Mitochondria (M) and RER appear normal. (x 28,900) B-An intracisternal Type A particle found in the same section as A (x 102,900).

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Figure 4A-This electron micrograph illustrates the spread of zymogen granule contents (arrows) through the exocrine cell cytoplasm (x 27,000). B-Lamellar membranous structure within lysed zymogen granule (x 23,900).

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FgV 5--A highly degenerated exocrine cell. The RER has become vesiculated and the mitochondna (M) swollen. Although many zymogen granules are completely digested (D), others (Z) appear unaffected. (x 25,000)

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Exocrine Pancreatic Insufficiency Syndrome in CBA/J Mice I. Ultrastructural Study John J. Eppig, PhD, and Edward H. Leiter, PhD The pathogenesis of a...
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