Clin. exp. Immunol. (1979) 36, 16-23.

Effect of sex hormones on the immune system ofguinea-pigs and on the development of toxoplasmic lesions in

non-lymphoid organs

C. KITTAS & L. HENRY Department ofPathology, University ofSheffield Medical School, Beech Hill Road, Sheffield

(Accepted frr publication 16 October 1978)

SUMMARY

The role of cell-mediated immunity in the pathogenesis of toxoplasmic lesions in non-lymphoidorgans and its significance in resistance to infection with Toxoplasma gondii were studied inguinea-pigs.

Alteration ofthe sex hormonal environment in both sexes has significant effects on the immuno-logical status of the guinea-pig, changing its reaction to toxoplasmic infection. Thus, gonadectomyappears to enhance delayed hypersensitivity, leading to a greater prominence of lesions in thenon-lymphoid organs of gonadectomized male and female animals than in controls. This obser-vation supports the possibility that delayed hypersensitivity plays a major role in the pathogenesisof non-lymphoid, toxoplasmic lesions. Hexoestrol administration for a long period of timeinduces complete thymic atrophy and severe involution ofthe thymus-dependent areas ofthe lymphnodes and spleen, leading to the suppression ofcell-mediated immunity and overwhelming diseasein those animals infected with Toxoplasma gondii, suggesting that cell-mediated immunity is ofimportance in resistance to toxoplasmic infection.

In view of these findings, the difficulties in the treatment of non-lymphoid toxoplasmiclesions, such as ocular ones, by depressing cellular immunity, are discussed.

INTRODUCTION

A process of hypersensitivity has been implicated in the pathogenesis of the toxoplasmic lesions in non-lymphoid organs by several investigators (Frenkel, 1948; Henry et al., 1973). However, it has also beensuggested that cellular immunity is of greater importance than the presence of circulating antibody inresistance to toxoplasmic infection (Frenkel, 1960; Stahl, Matsubayashi & Akao, 1966; Lindberg &Frenkel, 1977).Both androgens and oestrogens have been found to prevent corneal and skin graft rejections (Walt-

man, Burde & Berrios, 1971; Castro, 1974b) and to depress other reactions of delayed hypersensitivity(Kappas, Jones & Roitt, 1963; Mueller & Kappas, 1964). These observations reflect the depressiveeffect of sex hormones on cellular immunity. On the contrary, oestrogens induce an increased antibodyresponse to many antigens (Eidinger & Garrett, 1972; Thanavala, Rao & Thakur, 1973; Rangnekar, Rao& Joshi, 1974). An age-sex relationship has been found among British patients with toxoplasmiclymphadenopathy (Beverley et al., 1976). Moreover, histological differences in the appearances of thelymph node post-capillary venules of both control animals and those infected with Toxoplasma gondiihave been described (Henry & Beverley, 1976). These differences have been shown to be under sexhormonal influence (Kittas & Henry, 1978).

Correspondence: Professor L. Henry, Department of Pathology, University of Sheffield Medical School, Beech Hill Road,Sheffield SlO 2RX.

0099-9104/79/0040-0016$02.00 C 1979 Blackwell Scientific Publications

16

Toxoplasmic lesions in non-lymphoid organs 17In view of the above observations, an experiment was performed to study the effect of gonadectomy

and oestrogen administration on the development of lesions in non-lymphoid organs of guinea-pigsfollowing infection with Toxoplasma gondii.

MATERIALS AND METHODS

The experiments involved forty-eight mature guinea-pigs. Eight male and eight female animals were used as controls. Eightguinea-pigs of each sex were subjected to gonadectomy and in eight more, gonadectomy was subsequently followed byimplantation of a pellet containing Hexoestrol. These groups were then divided into two subgroups of four animals each, onebeing infected with Toxoplasma gondii, the other being left uninfected.Orchidectomy was performed under Althesin anaesthesia through a small transverse suprapubic incision; oophorectomy

was performed through bilateral lumbar incisions. Immediately following gonadectomy, half of the male and female guinea-pigs received a subcutaneous implant of a pellet containing 12 mg Hexoestrol ('Capomatic' Tablet S4B, Boots Co. Ltd.,Nottingham) through a small incision behind the left ear.

Inoculations. Four weeks after operation the animals were inoculated with a low virulence, cyst-forming strain of Toxoplasmagondii (Beverley strain). The inoculum was prepared from the brains of mice 9 weeks after infection. Fifty toxoplasma cystswere given subcutaneously as a diluted brain emulsion.

Examinations. Six weeks after infection, all the guinea-pigs were weighed and killed by exsanguination via the axillaryvessels under light ether anaesthesia. Portions of spleen, lymph node, thymus, lung, liver, heart, skeletal muscle and brainwere fixed in formal saline. The tissues were processed routinely to paraffin, sections cut at 40 pm and stained with haemat-oxylin-eosin, methyl-green pyronin and periodic acid-Schiff stains. The spleen and thymus were weighed before fixation on asensitive electronic balance. The posterior right quarter of the brain of each guinea-pig was emulsified with isotonic sterilesaline and examined as a wet unstained film for the presence of toxoplasma cysts. Using a cyst-counting system the number ofcysts per quarter of guinea-pig brain was estimated.

Antibody tests. These were done by a modification of the dye test of Sabin & Feldman (1948).Statistics. The differences were analysed statistically by the Student's t-test.Assessment oflesions. The severity of the lesions in the heart, skeletal muscle, brain, liver and lung were graded on the same

points basis previously used for the heart and skeletal muscle lesions in mice (Henry & Beverley, 1969): 0 = no lesions; 1 =minimal lesions; 2 = single areas or widespread minimal lesions; 3 = single moderately large lesions; 4 = single severe orwidespread moderate lesions.The sum of the points for each of the four animals in a group is the group score.

RESULTS

Two male and one female guinea-pig receiving Hexoestrol died 3-4 weeks after injection. Furtherguinea-pigs were used to restore the numbers in these groups.

In both sexes, gonadectomy was followed by a slight increase in body and spleen weight. The thymusweight ofoophorectomized (724.75+24-8 mg, mean+ s.d. ofthe mean) and orchidectomized (746+43 2)guinea-pigs was significantly higher than that of female (664 5+23 3 mg) and male (610+24 mg)controls, respectively (P<0-02 in females and P<0-01 in males). After Hexoestrol administration, afall in the body and spleen weights was seen in both male and female animals, while the thymus wasalmost completely atrophic.

Histology ofthe lymphoid organsWidening of the thymic cortex, the lymph node paracortex, and the white pulp of the spleen, mainly

round the central arteriole, was seen after gonadectomy of both male and female guinea-pigs. Theseareas were also more densely packed with lymphocytes than those of the controls.

After Hexoestrol administration, only remnants of thymic tissue were detected by cutting serialsections of the entire upper mediastinum. The thymic cortex was completely atrophic, only medullaryelements such as Hassall's corpuscles surviving. Numerous Foa-Kurloff cells were found in large num-bers in the thymic remnants and the spleen of both male and female guinea-pigs receiving Hexoestrol.The thymus-dependent areas of the spleen and lymph nodes, namely the area of the splenic white pulpround the central arteriole and the lymph node paracortex, were decreased in both sexes, while wideningof the lymph node medulla was also observed.

B

18 C. Kittas & L. HenryThe lymph node post-capillary venules showed similar but less prominent changes to those previously

described in mice under identical experimental conditions (Kittas & Henry, 1978).After infection with Toxoplasma gondii, the lymph nodes of the guinea-pigs of all groups showed

varying degrees of follicular reaction with germinal centre phagocytosis and varying numbers of plasmacells and epithelioid histiocytes. However, while a medullary reaction to infection was prominent incontrol male and female animals and even more prominent in Hexoestrol-treated ones, there was a greaterparacortical and a lesser medullary reaction when infection followed gonadectomy.The thymic changes after infection were minimal, ifany, in both control and gonadectomized male and

female guinea-pigs. The spleens of these groups showed prominent lymphoid follicles with germinalcentre phagocytosis and increased numbers ofplasma cells. Plasma cells were also increased in the spleensof infected animals that had received Hexoestrol, while there was atrophy of the white pulp with onlyminimal follicular reaction to infection.

Other organsThe total scores per group, obtained from the histological assessment of the lesions seen in the various

organs of the infected with Toxoplasma gondii guinea-pigs are shown in Table 1.

TABLE 1. Total score per group obtained by histological assessment of thelesions seen in non-lymphoid organs of guinea-pigs after infection with

toxoplasma

Histological score (maximum = 16)

SkeletalGroup Heart muscle Liver Lung Brain

Control:Male 4 2 1 1 1Female 4 2 1 2 0

Gonadectomy:Male 9 5 6 7 0Female 10 5 5 9 1

Gonadectomy and Hexoestrol:Male 8 1 5 4 0Female 9 3 5 5 0

Heart. The cardiac lesions after infection were minimal in both sexes (score 4 per each group). Thelesions consisted of a mild, focal, lymphocytic infiltrate and individual cell necrosis. Stellate areas offibrosis were occasionally seen, but no toxoplasma cysts were identified. Cysts were also absent in sectionsfrom the heart of gonadectomized, infected male and female animals, but the cardiac lesions were moreprominent than those of the infected controls (score 10 for the females and 9 for the males). Thus, theinflammatory infiltrate was more dense and although focal, it was more widely distributed throughout themyocardium. Subendocardial lesions were also seen.

In half of the infected male and female guinea-pigs receiving Hexoestrol toxoplasma cysts wereidentified in the myocardium. The cysts were unrelated to the inflammatory infiltrate present in onemale and two female animals. The infiltrate was diffuse and consisted mainly of plasma cells with only afew lymphocytes and polymorphs. The remaining three male and two female guinea-pigs in thesegroups showed no inflammatory reaction, despite the presence of toxoplasma cysts in the myocardiumof two of them. The score per group of the cardiac lesions was 9 for the females and 8 for the males.

Skeletal muscle. Mild changes of myositis were present in the muscle ofsome male and female infectedguinea-pigs (score 2 per each group). In both sexes, there was a slight increase in severity after gona-dectomy (score 5 per each group). A mild, mainly lymphocytic, interstitial infiltrate was seen in gonadec-

Toxoplasmic lesions in non-lymphoid organs 19tomized, infected animals (Fig. 1), unrelated to occasional toxoplasma cysts found within the musclefibres.

In Hexoestrol-treated animals, the number of cysts seen in the sections of muscle after infection wasincreased (Fig. 2). However, an inflammatory reaction was found in the muscle of only one animal, afemale, showing numerous cysts per section but only moderate insterstitial, mainly plasmacytic, infiltratenot related to the cysts present.

Liver. Minimal lesions only were seen in the liver of one male and one female infected control guinea-pig, the remainder showing no changes (score 1 per each group). On the contrary, all gonadectomizedanimals showed focal areas of lymphocytic infiltration either within the hepatic parenchyma or in theportal tract (score 6 for males and 5 for females). No toxoplasma cyst was found in any of the control orgonadectomized infected animals.

itertta infiltrae( E xk1_I607.)

Lung AlhuholAiia ein eese ntelugo netdcnrlaiaso ohsxs

Less dSensetbu moredffuseaiflamatorchinanpgeathan thoseirhoaectomizendinfectedaninlmhoyimas

itertiia iflrt. (H x> S160

AtrHxetoadiitaincytweefoundin oneivro wmale and one female (Fig.a-4).Brain.Threr mreanumesofteaicncoxopweelasma csspern goevrou foun infthemqartery breacionofstheinfected

animals aereacshowninTblp.)Thegnlhumbe ofncystsimadeceasednaferegoenaetm in btheugosnextesdacomparetol tatial of bthe inected,

controlnse,butaacsgnfculantioncese waslmhctseenweefonwhen infection followedHeostoamnitation.

(P<in0 T01) Occasinaiubesolated p cystswees eninsetons,fromnithe urebrain of theotoiadgnadecto-

anml resoninTbe2TheS nubro yt erae fe gndc yiohsxsa omae ota fteifcecotos bu a sinficntincrease was see whe infectio folowe Hexeo adiitraio.

FIG.00 1). OcSioneal,musolaed cystswerae seenine-i sectionfohreomtebano h otoandinetoghwnpondcyto

20 C. Kittas & L. Henry

FIG. 2. Skeletal muscle from a female guinea-pig after Hexoestrol treatment and infection showing threetoxoplasma cysts within the muscle fibres with no inflammatory reaction. (H & E x 400.)

Fig. 3. Lung from a male guinea-pig after orchidectomy and infection showing focal aggregates oflymphocytes.(H&E x160.)

21Toxoplasmic lesions in non-lymphoid organs

FIG. 4. Lung from a male guinea-pig, after Hexoestrol treatment and infection, and showing a Toxoplasmacyst (arrow). (H & E x 400.)

mized animals, while in several instances they were arranged in clusters in the Hexoestrol-treatedguinea-pigs. Minimal, if any, inflammatory changes were found in the meninges and the brain of theinfected animals of all groups.

TABLE 2. Mean numbers of toxoplasma cysts found in the quarter of the brain ofmale and female guinea-pigs infected with Toxoplasma gondii and subjected to

gonadectomy and administration of Hexoestrol

Sex Control Gonadectomy Gonadectomy and Hexoestrol

Male 82-5+ 21.1* 65 25+ 10-8 184+ 36-8Female 88-75+ 21-6 63-0+ 16-5 200 25+ 16

* +S.d.

SerologyThere was no significant difference between the groups and the average antibody titre ranged from

1/12,000 to 1/14,000.

DISCUSS IONAlteration of the sex hormonal milieu in both male and female guinea-pigs induces changes in both theseverity and character of the lesions in non-lymphoid organs, caused by toxoplasmic infection.Denser and more widely distributed lymphocytic aggregates were seen in the heart, skeletal muscle,

liver and lung of male and female gonadectomized guinea-pigs, 6 weeks after infection with Toxoplasmagondii, as compared with those of the infected controls. However, there were less cysts in the brains ofinfected gonadectomized animals than in controls.

22 C. Kittws & L. HenryIt has been shown that gonadectomy increases both thymic (Dougherty, 1952; Vasilakis, Kunz &

Gill, 1974) and splenic weight (Streng & Nathan, 1973), and also induces enlargement of the lymphnodes (Castro, 1974a). Our findings are in agreement with these observations. Histologically, there wasa widening of the thymic lobules, the lymph node paracortex and the areas around the central arteriolesof the spleen, after gonadectomy in both sexes; these areas are generally accepted to be involved in cell-mediated immunity (Parrott & De Sousa, 1971). These findings suggest that cellular immunity should beimproved after gonadectomy in both male and female guinea-pigs and they confirm previous observations(Graff, Lappe & Snell, 1969).We believe, therefore, that the more prominent lesions seen after toxoplasmic infection in non-

lymphoid organs of gonadectomized, rather than control, animals, are due to enhanced delayed hyper-sensitivity induced by the gonadectomy. The present results support the possibility that delayed hyper-sensitivity may be a component in the pathogenesis of the toxoplasmic lesions in non-lymphoid organs.When Hexoestrol was given, complete atrophy of the thymus and involutionary changes of the lymph

node paracortex and white pulp of the spleen were seen. These findings are similar to those reported byothers in rats (Heim & Timiras, 1963) and mice (Reilly et al., 1967; Kalland, Fossberg & Forsberg,1978), and suggest a depressive effect of Hexoestrol upon cellular immunity. This has also been confirmedby a depression of the reaction of delayed hypersensitivity seen after oestrogen administration (Kappaset a!., 1963; Mueller & Kappas, 1964; Ablin et al., 1974).When infection with Toxoplasma gondii followed Hexoestrol administration, the number of cysts

found in the brains of the infected guinea-pigs was increased; even in paraffin sections from variousorgans many toxoplasma cysts could be seen. This proliferation of cysts was not usually accompaniedby an inflammatory reaction. Inflammatory lesions in these animals, when present, were moderatelysevere but different in character to those seen in gonadectomized infected animals. Thus, the inflam-matory infiltrate in Hexoestrol-treated and later infected guinea-pigs was diffuse and contained mainlyplasma cells with few lymphocytes and polymorphs, in contrast to the focal, mainly lymphocytic, aggre-gates seen in infected gonadectomized animals. Interstitial oedema and occasional larger areas of necrosiswere also seen. Moreover, increased mortality was found in Hexoestrol-treated guinea-pigs when infectedwith Toxoplasma gondii. These results were seen despite the presence of high circulating antibody levelswhich were not significantly different from the titres found in all the other infected groups.We believe that the increased mortality and the other signs of overwhelming toxoplasmosis seen in

Hexoestrol-treated animals after infection, are due to a severe depression of cell-mediated immunitycaused by the constant effect of high doses of Hexoestrol on the thymus and the thymus-dependent areasof the lymph nodes and spleen for a long period of time. Similar findings have been reported by Frenkel(1960) and Stahl et al. (1966) on the impairment of the cellular immune system by corticosteroids. Theeffect of oestrogens on the thymus and the peripheral lymphoid tissues is not mediated by the adrenalsor the pituitary (Friedman et al., 1964; Pereira-Luz et al., 1969; Kalland et a!., 1978).Our observations, along with those made by Lindberg & Frenkel (1977) in mice, suggest that while

active cellular immunity is important in resistance to toxoplasmic infection, the role of circulating anti-body is uncertain.

The present findings support the possibility that cellular immunity plays a major role in the patho-genesis of the lesions in non-lymphoid organs. However, strong suppression of cellular immunity maylead to an overwhelming toxoplasmosis and death. Thus, although treatment of non-lymphoid toxo-plasmic lesions by depressing cell-mediated immunity may have beneficial results, such as the effect ofsteroids on ocular toxoplasmic lesions (Perkins, 1958; 1961), a critical point must not be exceeded beyondwhich resistance to the infection could be decreased.

This study was financed by a grant from the Medical Research Fund of Sheffield Uni% ersity. Thanks are due to Mr A. Ridgefor technical, Miss E. Hogg for secretarial and Mr M. J. Eaton for photographic assistance. The Hexoestrol u as obtained x iththe help of Mr D. A. Atkin, Boots Company Ltd., Nottingham.

Toxoplasmic lesions in non-lymphoid organs 23

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