American Journal ofPathology, Vol. 136, No. 5, May 1990 Copyright © American Association of Pathologists

Eosinophilic Myocarditis in CBA/J Mice Infected with Toxocara canis

Michael Cookston,* Martha Stober,t and Stephen G. Kayest From the Departments ofPatbology and Structural and Cellular Biology,t College ofMedicine, University ofSouth

Alabama, Mobile, Alabama

In humans, chronic eosinopbilia has been associated clinically with endomyocardial fibrosis and myocardial damage. Mice infected with Toxocara canis have a marked eosinophilia, and develop eosinophil-rich granulomatous lesions in the soft tissues of the body, especially the lungs, liver, brain, and skeletal muscle. Few reports have described myocardial lesions associated with T. canis infections in mice. We examined the hearts of CBA/J mice killed at weekly intervals over an 8-week periodfor evidence of myocardial damage that might be attributable to eosinophils. Total white blood cell counts and eosinophil counts were obtained during this period, and revealed a peak white blood cell count of approximately 28,000 cells/mm3 at day 7 after infection and a peak eosinophil count of approximately 4,000 cells/mm3 at day 14 after infection. Myocardial lesions in the ventricular wall began as focal infiltrates of eosinophils and histiocytes, then progressed into granulomata containing necrotic debris. Collagen deposition was noted by day 21 after infection. By day 42 after infection, the lesions had contracted greatly because of a loss of cellularity, and consisted mainly offibroblasts and hemosiderin-laden macrophages. Myocyte damage, characterized by increased eosinophilia and necrosis, was observed. T. canis-infected CBA/J mice thus offera useful model to study

eosinophil-dependent myocardial damage. (Am J Pathol 1990, 136:113 7-1145)

Chronic eosinophilia has been associated clinically with endomyocardial fibrosis and myocardial damage.' Chronic eosinophilia can be due to parasitic infection, such as visceral larva migrans (VLM), allergic disorders, drug reactions, or other causes. Dramatic eosinophil counts, as high as 90%, may occur in patients with VLM,

who often present with leukocyte counts of 30,000 to 100,000 white blood cells per cubic millimeter.23 Visceral larva migrans results from the ingestion of embryonated eggs of Toxocara canis or T. cati, the common roundworm of dogs and cats, respectively. After ingestion, hatching occurs in the proximal small intestine. The larvae penetrate the mucosa and migrate to the liver via the portal circulation, travel to the lungs, and enter the systemic circulation. When the size of a larva exceeds the blood vessel diameter, it may bore through the vessel wall of the host, migrate within the tissues, and elicit an immune response. Eventually, a larva becomes enmeshed in a fibrotic encapsulated granuloma.4'5'6 Severe myocarditis ensues when the second-stage larvae (L2) invade cardiac tissue. Several cases of myocarditis associated with VLM syndrome have been reported.37-9 Myocarditis may occur in 10 to 15% of cases of VLM, and in all cases, the myocarditis is accompanied by significantly increased levels of circulating eosinophils. Both mice and humans develop VLM after ingestion of T. canis ova.1014 The murine model of human VLM syndrome has been used previously15-17 to study the immunopathogenesis of pulmonary granuloma formation. We report herein that CBA/J female mice develop a severe eosinophilic myocarditis and describe the sequence of histopathologic changes and concomitant hematologic findings occurring in these mice during the first 56 days of infection.

Materials and Methods Mice Female CBA/J mice were obtained as weanlings (approximately 12 g) from the Jackson Laboratories (Bar Harbor, ME) and maintained on food and water ad libitum in the Animal Care Facilities of the University of South Alabama Supported by a grant from the American Heart Association, Alabama Affiliate (870034) and the National Institutes of Health (Al19968). Accepted for publication January 4, 1990. Address reprint requests to Stephen G. Kayes, PhD, Department of Structural and Cellular Biology, University of South Alabama, College of Medicine, Mobile, AL 36688.

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College of Medicine. These facilities meet all criteria for research animals as described in The Guide for the Care and Use of Laboratory Animals as promulgated by the National Institutes of Health. All procedures involving laboratory animals were approved by the Institutional Animal Care and Use Committee (IACUC). Mice were allowed to adapt to vivarial facilities for at least 7 days before being included in any experimental study.

Results Hematologic Findings Total WBC (Figure 1) were highest at 7 days after infection (28,000 cells/mm3) and returned to within normal limits by day 28. Eosinophils peaked at 20% to 25% of the total WBC by 14 days after infection (4000 cells/mm3) and declined to 15 to 20% during the remaining period of observation (Figure 2).

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Normal Myocardium Procedures for working with Toxocara canis have been described previously.15-17 Briefly, 250 infective ova were administered by gastric intubation of mice under light ether anesthesia.

Hematologic Studies Freely flowing tail vein blood was collected onto paraffin film and smeared onto cleaned microscope slides. The blood was diluted with Discombe's fluid,18 and the total number of eosinophils was counted using a hemocytometer. The blood smears were stained with a modified Wright's stain (Hemal-Stain, Hemal Stain Co., Inc., Danbury, CT), and 100 leukocytes differentiated per smear. Total leukocytes were counted using a Coulter Counter (Coulter Electronics, Hialea, FL). Before beginning these studies, all mice had white blood cell counts (WBC) of 10,000 cells or less and total eosinophil counts of 400 or less.

Histopathologic Studies Necropsies were performed on groups of four to six mice on days 7, 14, 21, 28, 35, 42, 49, and 56 (after infection). Hearts were removed and immediately fixed in Carson's formalin. All collected tissues were routinely processed, embedded in paraffin, sectioned at 4 to 6 A, and stained with hematoxylin and eosin (H & E) or Masson's trichrome. Three to four age-matched, uninfected litter mates were used as controls for the entire experiment. An average of six sections were made of each mouse heart and were examined microscopically. The number of lesions in the epicardium, myocardium, and endocardium were counted. The size of representative lesions was measured with an ocular micrometer, and the cell types constituting representative lesions were differentiated and counted.

Normal mouse myocardium (Figure 3) resembled normal human myocardium and could be easily distinguished from hearts of T. canis-infected mice.

Early Histologic Changes in Myocardium The heart at 7 days after infection showed linear infiltration of cells between the myocytes focally within the myocardium (Figure 4). These infiltrates consisted of equal numbers of eosinophils and histiocytes/lymphocytes. No evidence of myocardial damage could be demonstrated. No T. canis larvae were seen. The lesions measured approximately 0.11 mm in diameter. Approximately 10 lesions were seen per heart, with the majority (70%) of the lesions occurring in the myocardium. By 14 days after infection, larger, rounded granulomata were observed. Increased eosinophilia of the surrounding myocardial fibers was present. Portions of a single larva was observed in a single mouse heart (Figure 5). Early abscess formation with nuclear debris and myocyte fragments was observed with the granulomata (Figure 6). The granulomata averaged 0.38 mm in diameter and consisted of nearly equal numbers of eosinophils and histiocytes/lymphocytes. Approximately 11 lesions were seen per heart, and the lesions were equally distributed among the epicardium, myocardium, and endocardium. The granulomata at 21 days after infection were round with pushing borders. They contained a central abscess consisting mostly of necrotic debris. Prominent, increased eosinophila of the surrounding myocardial cells was observed. Trichrome stains revealed early peripheral fibrosis of the granulomata. No larvae were seen at this time in any of the hearts examined. Lesions measured approximately 0.35 mm in diameter. At 28 days after infection, the cellular composition of the granulomata had shifted to one third eosinophils and two thirds histiocytic cells. The size of the lesions had not changed from day 21 after infection, although they now contained a few multinucleated giant cells. Mild increased

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Figure 1. Total uhite blood cell (WBC) couints of uninfected conitrols (CTRL) and rnice inifected (INF) uith Toxocara canis dulring an 8-ueekperiod. Eachpoint is the miiean + 1 standard deviation of three

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eosinophilia of the cardiac myocytes was observed near the periphery of the granulomata. Some necrotic debris was present within the granulomata. Many histiocytes contained a light brown pigment within their cytoplasm, which stained positively for iron with a Prussian blue stain. Trichrome stain revealed a prominent peripheral fibrosis.

Late Histologic Changes in Myocardium Day 35 after infection was characterized by histiocytes and lymphocytes (86%) with a few eosinophils (14%). Prominent hemosiderin pigment deposition was seen

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within the histiocytes. No evidence of necrosis or increased eosinophilia of myocardial cells was noted. Trichrome staining revealed prominent fibrosis of the granulomata (Figure 7). The granulomata were much smaller at this time, averaging 0.14 mm in diameter. At day 42 after infection, the lesions averaged 0.12 mm in diameter and contained much fewer cells (96% histiocytes, many with prominent hemosiderin pigmentation). The granulomata were fibrotically encapsulated and no necrosis was observed. During the last 2 weeks of the study, the granulomata were difficult to find in H & E-stained sections, but were easily seen with trichrome staining. Lesions generally contained fewer than 30 cells

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Figure 4. Photomicrograph of mouse myocardium 7 days after infection with Toxocara canis. The myocardium contains smallfocal infiltrates of eosinophils and histiocytes (H&E X 160).

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Figure 5. Photomicrograph of mouse myocardium 14 days after infection withToxocara canis. Tangential and cross-sectionalprofiles ofan apparently viable second-stage larva are seen with minimal associated inflammatory reaction (H&E X400).

in a cross-sectional profile, and all were histiocytic. Lesions at the end of the study were 0.05 mm in diameter on average. In addition to the 8-week course of granuloma formation just described, new granulomata of varying ages were observed in the mouse heart during most of the later times sampled, indicating continuing larval migration. Larvae were rarely seen within the myocardium or the granulomata. Lesions associated with T. canis larvae showed no preference for any specific myocardial site, as lesions were seen within the epicardium, myocardium, and endocardium. Only an occasional mouse heart was free of any granulomata. In each of these cases, however, lesions were seen in other organs. One interesting finding was that the majority of the cardiac lesions were associated with a blood vessel. There was no evidence of diffuse endothelial or endocardial fibrosis. Granuloma formation with fibrotic encapsulation of the juvenile worms in sites other than the heart resembled lesions previously described by ourselves14 and others.10 Figure 8 depicts a developing granuloma at 42 days after infection around a T. canis larva situated in skeletal muscle removed from the nape of the neck. This lesion contains regions of acute reactivity (polymorphonuclear neutrophils [PMNs] and eosinophils) surrounded by areas of epithelioid macrophages and an occasional multinucle-

ated giant cell. This granuloma is compressing the adjacent skeletal muscle and somewhat resembles the lesions observed in the heart at day 28 after infection.

Discussion We have demonstrated that some of the larvae invade the myocardium and a severe eosinophilic myocarditis results after infection of CBA/J mice with Toxocara canis. This inflammation resolves with severe myocardial fibrosis, which sets in during the latter part of the third week after infection and continues to accrue through the eighth week of infection. Few, if any, larvae were observed in the heart after this time. Friedman and Hervada7 described a child with strong evidence of toxocaral infection who suffered from severe myocarditis that resolved on treatment. In contrast, Dent et al3 reported a myocardial lesion associated with a toxocaral fragment from a pediatric autopsy. The cellular composition of this lesion consisted primarily of lymphocytes, plasma cells, and only occasional eosinophils. Other sections of this heart showed only focal areas of interstitial myocarditis, with lymphocytes and macrophages predominating. The predominant cell observed in our experimental infections was the eosinophil. Thus, the heart le-

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Figure 6. Pbotomicrograpb of mouse myocardium 14 days after infection with Toxocara canis. A developing granuloma with a central abscess is seen near the epicardial surface. The lresion contains eosinopbils, bistiocytes, and polymorpbonuclear leutkocytes (H&E X 160).

sions described in human cases do not seem to resemble the lesions observed in experimentally infected mice. The pathology of toxocariasis in the human heart is not well documented, however. The fact that Dent's case was fatal may indicate that immunopathologic mechanisms, which were operating in our murine model, were overwhelmed, and other histologic cell types were allowed to participate. Our findings showed the damage produced to the heart by the migrating larvae of Toxocara canis. In response to the invasion of the heart, a granulomatous reaction rich in eosinophils was elicited. Myocyte destruction also was observed within these areas. This destruction may result either from damage caused by the migrating larvae or the inflammation and repair that follows. Several workers' 19-22 have described pathologic lesions in the heart attributable to large numbers of circulating eosinophils. Our hypothesis in performing these studies was the belief that mice infected with Toxocara canis would have large numbers of circulating eosinophils,1517'23 and, like patients with hypereosinophilia, would develop eosinophilic myocarditis. We did not expect to find large numbers of eosinophils infiltrating the heart, because few

workers have described eosinophil-rich myocardial lesions in murine or human toxocariasis. We believe that the damage to the myocardium seen in this study was the direct result of the inflammation incurred secondary to the toxocaral infection and was attributable to the large number of eosinophils infiltrating the heart. It also is possible that the myocardial damage seen was due to blood vessel obstruction by migrating larvae or possibly attributable to the larvae themselves. However, because few larvae were observed in the sections examined and because the amount of damage relative to the numbers of larvae seen was quite substantial, these latter possibilities do not seem likely. Mechanistically, mice infected with T. canis have high levels of gamma E immunoglobulin (IgE) (Finkelman, personal communication, 1989, unpublished results), and most likely have increased expression of surface Fc, receptors on their eosinophils to accommodate the circulating IgE. There is a substantial circulating antigenemia in T. canis-infected mice." If the toxocaral antigens were to bind to the T. canisspecific antibodies occupying these low-affinity Fc, receptors on circulating eosinophils, this would lead to eosinophil degranulation.25 If eosinophil degranulation occurred

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Figure 7. Photomicrograph of mouse myocardium 35 days after infection with Toxocara canis. Areas of marked fibrosis are seen surrounding several small intramyocardial veins. The lesions are less cellular, consisting mainly of histiocytes with a few lymphocytes and eosinophils (Masson 's Trichrome X 400).

intravascularly, then the cationic proteins would be expected to adsorb onto endothelial cells and lead to vascular insults. If degranulation occurred within the tissues, then the eosinophil proteins might absorb to the negatively charged myocytes and mediate intramyocardial lesions such as were found in this study. There is evidence for the latter possibility.21 The granules of the eosinophil stain avidly with acidic dyes and have a distinctive ultrastructure by electron microscopy.' Of interest here are the specific or secondary granules, which possess an electron-dense core surrounded by a lucent matrix. These granules contain a series of cationic proteins, including major basic protein (MBP), which makes up more than 50% of the dense core, and eosinophil peroxidase (EPO), which has been localized to the granule matrix in most species studied.29 Recent studies have suggested that eosinophils may play a cytotoxic role in a variety of diseases. Because all the eosinophil specific granule proteins are so cationic, they have a great affinity for coating negatively charged cell surfaces. It was hoped that this study would demonstrate that mice infected with T. canis would develop eosinophilic endomyocardial disease attributable to the marked eosinophilia that develops in toxocariasis. However, we found no evidence of diffuse endomyocardial damage or endo-

cardial fibrosis as described by Spry,1'20 who described myocardial lesions in patients with eosinophilic endomyocarditis and tropical endocardial fibrosis. Eosinophilic endomyocardial disease (EED) manifests itself as a layer of inflammation and fibrous tissue in the ventricles of the heart in patients with hypereosinophilia.26 Eosinophilic endomyocardial disease has been the focus of much research because it is a major complication of the hypereosinophilia syndrome (HES)2728 and can result in death from heart failure, thromboembolic disease, or arrhythmias. Recent evidence suggests that the eosinophil itself may be responsible for the observed cardiac lesions.19 Brockington and Olsen26 noted that eosinophilic endomyocardial disease occurs in three stages: the acute necrotic stage, the thrombotic stage, and the late fibrotic or end stage. The acute necrotic stage is seen in patients ill for only a few weeks, and is characterized by areas of acute necrosis and inflammatory cell infiltrates into the endomyocardium, with focal areas of damage to the subendocardium. After several months, the second stage begins when a layer of granulation tissue develops between the endocardium and subendocardium, accompanied by intraventricular thrombi and thrombi in small and mediumsized blood vessels. The end stage occurs when dense endomyocardial fibrosis is seen that produces a restric-

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Figure 8. Photomicrograph ofskeletal muscle 42 days after infection with Toxocara canis. A well-established grantuloma with central abscess containing longitudinal and cross-sectional profiles of a single L2 T. canis larva. This lesion resembles the intramyocardial lesions seen at 28 days after infection (H&1E x400).

tive myocardiopathy. The heart muscle hypertrophies because of restriction of systole and is often associated with incompetent valves, which result from the involvement of the papillary muscles by the inflammation, with replacement of myocardium by collagen. While our lesions occur mostly in the myocardium, they do for the most part seem to recapitulate the aforementioned sequence described in the endocardium of patients with EED, except for the development of granulation tissue with thrombus formation. Our lesions were focal however, and resolved with significant, but focal, myocardial fibrosis. No evidence of diffuse endocardial fibrosis was observed. Thus, we believe that female CBA/J mice infected with T. canis offer a useful animal model to examine the interactions of eosinophils and myocardium.

References 1. Spry CJF: Eosinophils and endomyocardial fibrosis: A review of clinical and experimental studies, 1980-86, Pathogenesis of Myocarditis and Cardiomyopathy. Recent Experimental and Clinical Studies. Edited by C Kawai, WH Abelmann. Tokyo, University of Tokyo Press, 1987, pp 293-310

2. Beaver PC, Snyder CH, Carrera GM, Dent JH, Lafferty JW: Chronic eosinophilia due to visceral larva migrans. Report of 3 cases. J Pediatr 9:7-19,1952 3. Dent JH, Nichols RL, Beaver PC, Carrera GM, Staggers RJ: Visceral larva migrans with a case report. Am J Pathol 32:

777-803,1956 4. Glickman LT, Schantz PM: Epidemiology and pathogenesis of zoonotic toxocariasis. Epidemiol Rev 3:230-250,1981 5. Schantz PM, Glickman LT: Roundworms in dogs and cats: Veterinary and public health considerations. Cont Educ Prac Vet 3:773-784,1981 6. Schantz PM, Glickman LT: Toxocaral visceral larva migrans. N Engl J Med 298:436-439,1978 7. Friedman S, Hervada AR: Severe myocarditis with recovery in a child with visceral larva migrans. J Pediatr 56:91-96,

1960 8. Brill R, Churg J, Beaver PC: Allergic granulomatosis associated with visceral larva migrans. Case report associated with autopsy findings of Toxocara infection in a child. Am J Clin Pathol 23:1208-1215,1953 9. Vargo TA, Singer DB, Gillette PC, Fernbach DJ: Letters to Editor. Myocarditis due to visceral larva migrans. J Pediatr 90:322-323,1977 10. Burren CH: Experimental toxocariasis. l. Some observations on the histopathology of the migration of Toxocara canis. Z Parasitenkund 30:152-161, 1968

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11. Sprent JFA: On the migratory behavior of the larvae of various Ascaris species in white mice: I. Distribution of larvae in tissues. J Infect Dis 90:165-176,1952 12. Dunsmore JD, Thompson RCA, Bates IA: The accumulation of Toxocara canis larvae in the brains of mice. Int J Parasitol 13:517-521,1983 13. Olson LJ: Organ distribution of Toxocara canis larvae in normal mice and mice previously infected with Toxocara, Ascaris or Trichinella. Tex Rep Biol Med 20:651-657,1962 14. Kayes SG, Oaks JA: Development of the granulomatous response in murine toxocariasis. Initial events. Am J Pathol 93:

277-294,1978 15. Kayes SG: Spleen cell responses in experimental murine toxocariasis. J Parasitol 70:522-529, 1984 16. Kayes SG, Omholt PE, Grieve RB: Immune responses of CBA/J mice to graded infections with Toxocara canis. Infect Immun 48:697-703,1985 17. Kayes SG, Jones RE, Omholt PE: Use of bronchoalveolar lavage (BAL) to compare local pulmonary immunity with the systemic immune response in mice infected with Toxocara canis. Infect Immun 55:2132-2136,1987 18. Discombe G: Criteria of eosinophila. Lancet i: 195-196,1946 19. Olsen EG, Spry CJF: The pathogenesis of Loffler's endomyocardial disease and its relationship to endomyocardial fibrosis. Prog Cardiol 8:281-303,1979 20. Spry CJF, Davies J, Tai P-C, Fattah D: The pathogenesis of eosinophilic myocardial disease, Immunobiology of the Eosinophil. Edited by T Yoshida, M Torisu. New York, Elsevier Science Publishing Company, pp 229-244,1983 21. Tai P-C, Hayes DJ, Clark JB, Spry CJF: Toxic effects of human eosinophil secretion products on isolated rat heart cells in vitro. Biochem J 204:75-80,1982

22. Tai P-C, Spry CJF, Olsen EG, Ackerman SJ, Dunnette S, Gleich GJ: Deposits of eosinophil granule proteins in cardiac tissues of patients with eosinophilic myocardial disease. Lancet i:643-647,1987 23. Kayes SG, Oaks JA: Toxocara canis: Role of the T lymphocyte in murine visceral larva migrans and its relationship to onset of eosinophilia. Exp Parasitol 79:47-55,1980 24. Bowman DD, Mika-Grieve M, Grieve RB: Circulating excretory-secretory antigen levels and specific IgG and IgM responses in mice infected with Toxocara canis. Am J Trop Med Hyg 36:75-82,1987 25. Capron M, Bazin H, Joseph M, Capron A: Evidence for IgEdependent cytotoxicity by rat eosinophils. J Immunol 126: 1764-1768,1981 26. Brockington IF, Olsen EG: Lofler's endocarditis and Davies' endomyocardial fibrosis. Am Heart J 85:308-322,1973 27. Chusid MJ, Dale DC, West BC, Wolff SM: The hypereosinophilic syndrome. Analysis of 14 cases with review of the literature. Medicine (Baltimore) 54:1-27,1975 28. Parrillo JE, Borer JS, Henry WL, Wolff SM, Fauci AS: The cardiovascular manifestations of the hypereosinophilic syndrome. Am J Med 67:572-582,1979 29. Gleich GJ, Loegering DA: Immunobiology of eosinophils. Ann Rev Immunol 2:429-459,1984

Acknowledgments The authors thank Drs. William Gardner and Thomas L. Deardorff for critical reading of the manuscript.

J mice infected with Toxocara canis.

In humans, chronic eosinophilia has been associated clinically with endomyocardial fibrosis and myocardial damage. Mice infected with Toxocara canis h...
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