605

SPONTANEOUS AND EXPERIMENTAL MYOCARDITIS:THE EFFECT OF FREUND'S ADJUVANT ON THE HEART

AND OTHER ORGANS*

A. LAUFER, E. ROSENMANN AND A. M. DAVIESFrom the Rothschild Hadassah University Hospital, Departments of Pathology

and Medical Ecology, Hebrew University-Hadassah Medical School,Jerusalem, Israel

Received for publication May 27, 1966.

FREUND'S mineral-oil mixture and its variations are widely used as adjuvantsin the stimulation of antibody response to a variety of antigens (Freund, 1951;Kaplan, 1958; Kaplan and Craig, 1963; Brown, Glynn and Holborow, 1963;Laufer, Ginsburg, Gery and Davies, 1963; Davies, Laufer, Gery and Rosenmann,1964; and Cuppage, 1965). That the mycobacterial adjuvant by itself canproduce a variety of pathological responses, ranging from arthritis to generalizeddisease in the rat (Pearson, 1956; Pearson, Waksman and Sharp, 1961; Pearsonand Wood, 1963) granulomatous lesions of the lungs (Amemori and Altschul,1963; Allegretti and Vitale, 1965) and of other organs (Thal, Laufer and Behar,1958; Laufer, Thal and Behar, 1959; Steiner, Langer and Schatz, 1960; Jahieland Koffler, 1961; Brown et al., 1963; Geduldig, Reubner and Iber, 1964) of avariety of animals, with, in mice, amyloid deposits, (Laufer et al., 1959; Thal,Laufer and Zlotnick, 1964) is well established.

The production of experimental myocarditis in laboratory animals by injectionof heart muscle extracts in Freund's complete adjuvant (Davies et al., 1964), theoccasional appearance of lesions in control animals and the prevalence of " spon-taneous " myocarditis in laboratory stocks, prompted this study of the effects ofthe adjuvant itself on the heart and other organs.

MATERIAL AND METHODSAnimals.-Outbred albino rats of the Hebrew University strain weighing 60-110 g.,

albino mice (35-40 g.) and pen bred guinea-pigs (170-370 g.) were used in these experiments.Mongrel rabbits (about 2-5 kg.) were obtained from a dealer and Syrian hamsters (90-120 g.)from the laboratories of the Beilinson Hospital. The numbers examined and the treatmentgiven are shown in Table I. Control animals, matched for weight but not for sex, werereceived at the same time as the test animals and were kept in adjacent cages receiving thesame diet but no treatment.

Freund's complete adjuvant.-(Difco) was emulsified with an equal volume of saline andinjected i.c. in the stated dosages divided between 2 sites, weekly for 6 weeks.

Method&.-One week after the last injection, the animals were killed by exanguinationfrom the neck vessels while under ether anaesthesia, except for the rabbits which were killedby a blow on the back of the neck. Immediately after death, whole organs, or samplepieces in larger animals, were removed and fixed in neutral formalin.

Paraffin sections of the organs cut at 10,u were stained with haematoxylin and eosin aswell as with special stains for amyloid where indicated.

* This work was supported by Research Grant HE-05396 from the U.S. National Institutes ofHealth.

A. LAUFER, E. ROSENMANN AND A. M. DAVIS

Sera were examined for the presence of anti-heart antibodies in the passive haemagglutina-tion test using human group 0 erythrocytes coated with extracts of homologous and hete-rologous heart (Gery and Davies, 1961).

RESULTSSerological findings

None of the animals, test or control, showed antiheart antibodies of a titrehigher than 1: 5, i.e. all sera were " negative " by definition (Gery and Davies,1961).Histopathological findings (Tables I and II)

Heart.-The histological changes in the myocardium were the same in treatedand untreated animals but varied in distribution and intensity in the different

TABLE I.-Study Design and Summary of ResultsNumber of animals with pathological lesions*

Treatment

Adjuvant-0. 5 ml.weekly for 6 weeksNone-controls

Adjuvant-0 5 ml.weekly for 6 weeks

None-controls

Adjuvant-0-5 ml.weekly for 6 weeks

None-controls

Adjuvant-0- 7 ml.weekly for 6 weeks

Heart Lungs

4/25 14/20

4/27 111220/15 10/14

0/8 8/85/44 35/42

3/31 20/2513/26 23/25

Spleen

20/24Liver

20/24

3/23 12/260/15 5/14

Pancreas andmesenteric

fat

15/22 5/25

0/210/14

1/8 1/8 0/80/40 8/42 4/43

0/2710/25

5/268/24

0/2813/19

14 . None-controls

* No. with pathological changesNo. animals examined

Number of

7/14 8/14 3/14 0/12 0/13

Fre,und's Adjuvant*Pancreas

andmesenteric

Species animals Heartt Lungs Spleen Liver fat KidneyiMouse. 25 . (4/25) . 1/20 . 12/244 . 14/24 . 15/22 0/25Hamster . 15 . 0/15 . 0/14 . 0/16 . 0/14 . 0/14 . 0114Rat . 44 . (5/44) . 3/42 . 0/40 2/42 . 4/43 . 0/42Guinea pig . 26 . (13/26) . 11/26 10/25 . 6/24 . 13/19 . 0/25

* Numbers with lesions/numbers examined.t See text. The number ofanimals with cardiac lesions were similar in treated and control animals.t Of the 12 affected mice, 3 showed presence of amyloid.

Guinea pig270 100 g.

Speciesand weight

Mouse37 2g.

Hamster105 + 15 g.

Rat .85 25g.

Number ofanimals

25

27

15

8

44

Kidneys

31

26

5/250/14

0/81/42

1/271/25

TABLE II.-Pathological Lesions Attributable to

1/12

606

8

SPONTANEOUS AND EXPERIMENTAL MYOCARDITIS

species. The characteristic feature was an interstitial infiltration of small ormedium sized round cells, usually at single, but sometimes multiple, foci or scatteredbetween the muscle fibres. In rats and guinea pigs, the infiltrate displaced, andsometimes replaced, the muscle fibres at some points (Figs. 1, 2, 3, 4, 5). None ofthe hamsters showed myocardial lesions while the rats were unusual in thattreated animals showed an interstitial infiltration deep between the myocardialfibres while in the control group, the infiltrate was subepicardial in position.

Foam cells were not seen in any of the preparations.ILungs.-Only mice, rats and guinea pigs which had received adjuvant, showed

granulomatous lesions (Fig. 6). The granuloma consisted of epitheloid cells witha few giant cells and foam cells, fat vacuoles surrounded by lymphoid cells beingpresent in some of them. No necrosis was present in the granulomata nor wereacid fast bacilli detected. The granulomata were single or conglomerated andsituated either in the parenchyma or near small vessels or bronchioles. Peri-vascular cuffing with lymphoid cells was seen in several of the rats and guineapigs.

Areas of congestion and haemorrhage and interstitial pneumonia were seen insome of the animals-both treated and not.

Liver.-The treated animals showed capsular and/or parenchymal changes ofvarying distribution and intensity. The capsular lesions consisted of granulomatawith macrophages and fat vacuoles (Fig 7). Similar granulomata were presentin the liver parenchyma of mice, rats and guinea pigs in varying degrees (Fig. 8).A round cell infiltration varying in intensity was present in the portal spaces orin the sinusoids extending into the parenchyma, sometimes accompanying granu-lomatous lesions (Fig. 9). The control group showed, rarely, a minimal roundcell infiltration in the portal spaces, but no granulomata.

Kidneys.-Interstitial round cell infiltrations were present in some of thetreated and untreated animals; there were no glomerular lesions.

Pancrea8.-Lesions in the pancreas and peripancreatic fat were seen in treatedmice, rats and guinea pigs. They consisted of round cell infiltration in theinterlobular septa and peripancreatic fat tissue with granulomata in the peri- andinter-pancreatic septa (Fig. 10). The granulomatous lesions consisted of epitheloidcells surrounded or mingled with foam cells or large fat vacuoles, round cellinfiltration and marked proliferation of fibroblasts (Fig. 11). Neutrophils wererare but there were many capillaries and distended vessels lined by flattenedendothelial cells. The pancreatic parenchyma was intact. The control animalsdid not show any changes in the pancreas.

Spleen.-Histological changes appeared only in mice and guinea pigs givenadjuvant and were present both in the splenic tissue and capsule. The follicles,in some animals, were large without clear boundaries and the red pulp showedmarked histiocytic proliferation. In the mouse spleen there were abundant giantcells and this species, alone, showed amyloidosis, of a perifollicular and/or pulpardistribution (Fig. 12). The amyloid showed characteristic properties, i.e. stainingwith PAS and Congo-red, metachromasia with gentian violet and dichroism inpolarized light.

While the splenic capsule was usually thin, local thickening was found in someanimals. This consisted ofround cell infiltration with fibroblasts, fibrocytes withincollagen fibres as well as foamy epitheloid cells, forming a granulomatous reactionwithout necrosis. Many dilated lymph vessels were present, filled with small

607

A. LAUFER, E. ROSENMANN AND A. M. DAVIS

lymphocytes and lined by one or two layers of elongated or cuboidal cells, here andthere accumulating into giant cells. In some places there were adhesions to theinflamed peri-pancreatic fatty tissue.

Splenic lesions, present in some of the control animals, were limited to anoccasional large follicle.

DISCUSSION

The histological changes in the hearts of injected animals and untreatedcontrols are morphologically identical but vary in intensity and distribution.The one exception, in the rat myocardium, lies in the localization of the cellularinfiltrate which is interstitial in treated animals, rather than subepicardial incontrols. The amounts of Freund's adjuvant received by these animals weremany times larger than in other series (Pearson, 1956; Pearson et al., 1961;Pearson and Wood, 1963) and thus one would have expected more or differentlesions in injected animals. Their similarity to the controls suggests that thelesions observed are those of " spontaneous myocarditis " although the possibilityof minimal damage by the adjuvant cannot be excluded. The results obtained inother organs by injection of adjuvant by different routes, alone and in combinationwith antisera (Roitt, Jones and Doniach, 1961; Geduldig et al., 1964; Watson,Dixon and Feldman, 1965; Cuppage, 1965) indicate a damaging effect of theadjuvant itself although leaving unsolved the precise mode of action.

" Spontaneous myocarditis " has been reported by several authors, usually inthe sense of myocarditis of unknown cause. Up to two-thirds of the apparentlyhealthy mice of different colonies, have been affected (Miller, 1924; Wilens andSproul, 1938a, b; Loewe and Lenke, 1940; Pearson and Wood, 1963) andwe have noted similar changes, though less frequently, in guinea pigs, rats andrabbits (Davies et al., 1964). The same histological picture is produced byendotoxin (Kovats, 1961; Davies, Gery, Rosenmann and Laufer, 1963) and

EXPLANATION OF PLATESFIG. 1. Rat-injected with adjuvant. Interstitial focus of myocarditis replacing

muscle fibres and consisting of small and medium sized round cells. H and E X 405.FIG. 2.-Guinea pig-injected with adjuvant. Interstitial focus of myocarditis, consistingof mainly small round cells mingled with some histiocytic cells. H and E X 396.

FIG. 3.-Rat, control group-Diffuse focus of interstitial myocardial infiltration.HandE x405.

FIG. 4.-Guinea pig, control group-small interstitial focus of infiltrationdisplacing the surrounding muscle fibres. H and E x 405.

FIG. 5.-Mouse intracutaneously injected with Freund's adjuvant.Small interstitial focus of myocarditis. H and E x 405.

FIG. 6.-Guinea pig-injected with Freund's adjuvant. Granulomatous lesionspresent in the lung parenchyma. H and E x 410.

FIG. 7.-Mouse-injected with Freund's adjuvant. Liver capsule, thickened, infiltratedwith numerous granulomata. Note the presence of dilated lymphatics. H and E X 38.

FIG. 8.-Mouse-injected with Freund's adjuvant. Granuloma present in the liverparenchyma. Note the presence of large fat vacuoles. H and E x 405.

FIG. 9.-Rat-injected with Freund's adjuvant. Round cell infiltration in the portalspace of the liver extending into the parenchyma. H and E x 100.

FIG. 10.-Mouse-injected with Freund's adjuvant. Peri-pancreatic fat granuloma;mild interstitial round cell infiltration. H and E x 405.

FIG. 11.-Mouse-injected with Freund's adjuvant. Fat granulomain the pancreatic septa. H and E x 405.

FIG. 12.-Mouse-injected with Freund's adjuvant.Diffuse amyloidosis in spleen. H and E x 405.

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SPONTANEOUS AND EXPERIMENTAL MYOCARDITIS

although no microorganisms were found in sections of the heart, the animalbreeding stocks suffered from endemic salmonellosis (mainly Salmonella typhi-murium) and these organisms in the gut might well have released endotoxin intothe circulation (Davies et al., 1963). Another aetiological possibility is virusinfection and coxsackie virus among others, may cause lesions indistinguishablefrom those seen (Moore, Ridge, Huntington, Hall, Riffith and Knowles, 1947;Laruelle and Reumont, 1952). The " spontaneous myocarditis " seen in therat is usually mild (Davies et al., 1963) while the hamster, in this and previousseries, was never affected. This fact emphasizes the differences in susceptibilityof different species and suggests the hamster to be a suitable animal for studieson experimental myocarditis. The guinea pig showed the greatest sensitivityto Freund's adjuvant as illustrated by the lesions present in lung, liver, pancreasand peripancreatic fat and splenic capsule. That this might be a reflection of thisspecies' known sensitivity to toxic and immunological stimuli is suggested by therelatively high prevalence of myocarditis in control as well as in treated animals.

It is likely that the adjuvant elicits the cellular reactions in one of two waysdepending on the amount and route of injection (Steiner et al., 1960; Dalle, 1960,1961; Schoenberg and Moore, 1961). Dispersal of the oil phase as microemboliwill produce direct tissue damage with resulting granuloma formation. Stimula-lation of the reticuloendothelial system in a way not fully understood butconnected with the mycobacterial fractions will result in a " hypersensitivity"reaction with many manifestations (Freund, 1951, 1957; Laufer et al., 1959;Pearson et al., 1961; Steiner et al., 1960; Pearson and Wood, 1963; Thal et al.,1964).

Thus the pancreatic lesions could result from damage of the cell membrane bycontact with the oil droplets or the mycobacterial antigen releasing proteolyticand lipolytic enzymes. Here again, the species difference was marked, lesions ofthe pancreas being frequent in guinea pigs and mice, rare in rats and absent inhamsters. Granulomata of lung, liver and pancreas are noted and have beenpreviously described (Freund, 1951 ; Thal et al., 1958; Laufer et al., 1964;and Geduldig et al., 1964) while Amemori and Altschul (1963) describe foam likecells in the myocardium after i.v. injection of adjuvant.

The hypersensitivity reactions described have been elicited with small dosesof mycobacterial adjuvant (Allegretti and Vitale, 1965) and microemboli were notfound in the vicinity of, for instance, arthritic joints (Pearson, 1956; Pearson et al.,1961; and Pearson and Wood, 1963). None ofhundreds ofsections ofmyocardiumexamined in this and previous series has shown foam cells or the presence ofadjuvant droplets adjacent to focal myocarditis. The resemblance between thelesions observed and those of the myocarditis of homologous disease (Stastny,Stembridge, Vischer and Ziff, 1965) suggest an immune mechanism. We havesuggested elsewhere however (Laufer and Davies, 1966) that in the heart, theeffects of " hypersensitivity ", as those of infection, endotoxin or anoxia, aremediated through a final common path, involving changes in cellular permeability,lysosome damage and autolysis and there is a mounting body of supportingevidence (Laufer and Davies, 1966; Gazenfeld, Rosenmann, Davies, and Laufer,1966). The absence of circulating antibodies is in accord with this suggestion(Davies, et al., 1963, 1964; Laufer and Davies, 1966).

It is, in fact, doubtful whether such an entity as " spontaneous myocarditis"exists and the lesions so described are most likely the standard histological response

609

610 A. LAUFER, E. ROSENMANN AND A. M. DAVIS

through different pathways to one or other of a variety of agents. The verymonotony of the cellular response (Laufer and Davies, 1966) demands the mostcritical analysis and the use of adequate controls, in all experiments involvinginterpretation of the lesions of experimental myocarditis.

SUMMARY

The effect of Freund's adjuvant on experimental myocarditis in differentspecies was studied in relation to " spontaneous myocarditis ". Treated animalsshowed lesions of lungs, spleen, liver and their serosal surfaces, pancreas andperipancreatic fat, that were attributable to the action of the adjuvant.Pancreatic lesions were more prominent in mice and guinea pigs, amyloidosis waspresent only in mice while hamsters showed no specific lesions indicating a speciessusceptibility.

The possible pathogenesis of " spontaneous myocarditis " was discussed in thelight of the probability that different causes mediate through a final commonpathway to produce a standard histological response.

REFERENCESALLEGRETTI, N. AND VITALE, B.-(1965) Immunol., 9, 11.AMEMORI, T. AND ALTSCHUL, R.-(1963) Zbl. allg. Path. path. Anat., 104, 352.BROWN, P. C., GLYNN, L. E. AND HOLBOROW, E. J.-(1963) J. Path. Bact., 86, 505.CUPPAGE, F. E.-(1965) Lab. Invest., 14, 514.DALLE, M. M.-(1960) Br. J. exp. Path., 41, 86-(1961) Br. J. exp. Path., 42, 297.DAVIES, A. M., LAUFER, A., GERY, I., ROSENMANN, E. AND LAUFER, A.-(1963) Proc.

Soc. exp. Biol. Med., 114, 520.DAVIES, A. M., GERY, I. AND ROSENMANN, E.-(1964) Arch. Path., 78, 369.FREUND, J.-(1951) Am. J. clin. Path., 21, 645.-(1957) J. Allergy, 28, 18.GAZENFELD, E., ROSENMANN, E., DAVIES, A. M. AND LAUFER, A.-(1966) Immunol.,

10, 193.GEDULDIG, M. M., REUBNER, B. AND IBER, F. L.-(1964) Gastroenterology, 46, 175.GERY, I. AND DAVIES, A. M.-(1961) J. Immun., 87, 351.JAHIEL, R. I. AND KOFFLER, D.-(1961) Br. J. exp. Path., 42, 338.KAPLAN, M. H.-(1958) J. Immun., 80, 254.KAPLAN, M. H. AND CRAIG, J. M.-(1963) J. Immun., 90, 725.KOVATS, T.G.-(1961) Naturiwissenschaften, 48, 572.LAUFER, A. AND DAVIES, A. M.-(1966) In " Methods and Achievements in Experimental

Pathology ", Vol. II, Ed. Bajusz, E. and Jasmin G. Basel: (Karger) (in press).LAUFER, A., GINSBURG, I., GERY, I. AND DAVIES, A. M.-(1963) Path. Biol., 11, 769.LAUFER, A., THAL, C. AND BEHAR, A. J.-(1959) Br. J. exp. Path., 40, 1.LARUELLE AND REUMONT-(1952) Ann. Inst. Pasteur, 83, 543.LOEWE, L. AND LENKE, S. E.-(1940) J. exp. Med., 71, 89.MILLER, P.-(1924) J. exp. Med., 40, 543.MOORE, J. F., RIDGE, G. K., HUNTINGTON, R. W., HALL, E. M., RIFFITH, G. ANI)

KNOWLES, R. G.-(1947) Proc. Soc. exp. Biol. Med., 65, 102.PEARSON, C. M.-(1956) Proc. Soc. exp. Biol. Med., 91, 95.PEARSON, C. M., WAKSMAN, B. H. AND SHARP, J. T.-(1961) J. exp. Med., 113, 485.PEARSON, C. M. AND WOOD, F. D.-(1963) Am. J. Path., 42, 73.ROITT, I. M., JONES, H. E. H. AND DONIACH, DEBORAH-(1961) In lInd International

Symposium on Immunopathology, Ed. Grabar, P. and Miescher, P., Stuttgart.(Benno Schwabe), p. 174.

SPONTANEOUS AND EXPERIMENTAL MYOCARDITIS 611

SCHOENBERG, M. D. AND MOORE, R. D.-(1961) Arch. Path., 72, 424.STASTNY, P., STEMBRIDGE, V. A., VISCHER, T. AND ZIFF, M.-(1965) J. exp. Med., 122,

681.STEINER, J. W., LANGER, B. AND SCHATZ, D. L.-(1960) Arch. Path., 70, 424.THAL, C., LAUFER, A. AND BEHAR, A. J.-(1958) Br. J. exp. Path., 39, 159.THAL, C., LAUFER, A. AND ZLOTNICK, A.-(1964) Br. J. exp. Path., 45, 323.WATSON, J. T., DIXoN, F. J. AND FELDMAN, J. D.-(1965) Lab. Invest., 14, 1559.WILENS, S. L. AND SPROUL, E. E.-(1938a) Am. J. Path., 14, 177-(1938b) Am. J. Path.,

14, 201.

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