Br. J. exp. Path. (1977) 58, 311

THYMUS DEPENDENCE AND INDEPENDENCE OF INTESTINAL PATHOLOGY IN A TRICHINELLA SPIRALIS INFECTION: A STUDY IN CONGENITALLY ATHYMIC (NUDE) MICE E. .J. lUITENBERG, F. LEENSTRA AND A. ELGERSMA From the Laboratory of Pathology, National Institute of l'ublic Health, P.O. Box 1, Bilth,oven, The Netherlands Received for publication February 1, 1976

Summary.-The hypothesis that non-specific histopathological gut changes are thymus-dependent was tested in a helminth infection-the nematode Trichinella spiralis. The study was performed in congenitally athymic (nu/nu) mice and their thymus-bearing heterozygous litter-mates (+/nu). The non-specific histopathological changes of the small intestine were judged on villus/crypt ratio and mitotic index; for the specific (= immunological) reaction the number of pyroninophilic cells in the small intestine was used as criterion. T. spiralis induced non-specific histopathological reactions both in nu/nu and +/nu mice, whereas the number of pyroninophilic cells was only increased in +/nu mice. It was concluded that the immunological reaction was dependent on the immune status of the host, whereas the non-specific histopathological changes were not, i.e. they were thymus-independent.

RECENTLY, Ferguson and Jarrett (1975) suggested non-specific histopathological gut changes due to a Nippostrongylu8 bra8iliensis infection in the rat to be thymus-dependent. Partial or sub-total villous atrophy with crypt hyperplasia occurred in affected areas of small intestine in normal rats, but this enteropathy was absent in 70%0 of experimentally infected rats which had previously been depleted of thymus-dependent (T) cells by thymectomy and irradiation. Since this observation could have a large impact on the understanding of intestinal pathology, we decided to verify this hypothesis by performing similar experiments in congenitally athymic (nude, nu/nu) mice and their thymus-bearing heterozygous litter-mates (+ /nu) using another helminth as the infectious agent. For this purpose we chose the intestinal tissue-invading nematode Trichinella spiralis. In +/nu mice T. spiralis adult worms are expelled from the gut within 10 days post infection (p.i.), whereas in nude mice 21*

worms are present until at least 83 days p.i. (Ruitenberg et al., 1977; Ruitenberg and Steerenberg, 1974), indicating T-cell dependence of host protection against a T. spiralis infection. MATERIALS AND METHODS

Male SPF B 10 LP nude mice, 6 weeks of age, were obtained from the Central Laboratory for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands, where the mice were maintained by conventional back-crossing with Strain B 10 LP. Comparisons were made with age-matched + /nu mice. At 8 weeks of age the mice were orally infected with 100 T. spiralis larvae according to previously published procedures (Ruitenberg and Steerenberg, 1974). T. spiralis-infected animals were examined at autopsy 3-16 days p.i. Non-infected controls were examined by autopsy at Day 0. The intestinal pathology was studied in a 10-cm portion of the proximal jejunum with a Swiss-roll technique (Reilly and Kirsner, 1965). The tissue was fixed in 4% formalin and embedded in paraplast. Sections were stained with haematoxylin and eosin for studying general morphological features. For specific

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staining of pyroninophilic (= immunocompetent) cells, including plasma cells, tissues were fixed in 90 parts Zenker's fluid, 5 parts commercial formalin (40%) and 5 parts trichloracetic acid (ZFT). Paraplast sections were stained with methyl green pyronine. Non-specific histopathological changes were judged on villus/crypt ratio and mitotic index (Swanson and Thomassen, 1965). For the

specific (= immunological) reaction the number of pyroninophilic cells including plasma cells was used as a criterion. Villus/crypt ratio was determined by measuring the length of 10 villus/crypt units per animal by means of a Zeiss Visopan microscope. The mitotic index in the crypts of Lieberkuhn was determined by counting a total number of 200-300 nuclei in approximately 10 crypts

FIGe. 1. Mucosa of jejunum of non-infected nu/nu mouse. Normal architecture with scalloping of epithelial lining. Paraplast section. H. and E. x 200. FIG. 2.-Mucosa of jejunum of nu/nu mouse 7 days after infection with 100 T. spiralis larvae. Villi are shortened and crypts of Lieberkuhn show hyperplasia. Distinct mitotic figures in epithelial cells of Lieberktihn crypts can be distinguished. Paraplast section. H. and E. x 200.

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TRICHINELLA SPIRALIS IN ATHYMIC MICE

per animal. The number of mitoses was expressed per 100 epithelial crypt cells. The number of pyroninophilic cells in the intestinal mucosa was counted per 20 villus/ crypt units. A villus/crypt unit represents a portion of gut inucosa, lying between two gland crypts and the lamina propria of the villus above (.Jarrett et al., 1968).

T.spiralis number of pyroninophilic cells

24F_ 22_

20F_ RESULTS

The morphology of the mucosa of the jejunum of non-infected +/nu and nu/nu mice was similar. In both cases villi were long and slender, showing distinct scalloping of the epithelial lining. The ratio between villus length and depth of the crypt was approximately 3: 1 (Fig. 1). After infection with T. spiralis the villus/crypt ratio in both

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Mitotic index (number of mitoses epithelial crypt nuclei) and villus/crypt ratio (mean of 10 villus/crypt units) of the proximal jejunum in +/nu or nu/nu mice at various days after oral infection with 100 T. spiralis larvae (mean values ± s.e. mean of groups of 4 per

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groups of 4 animals

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FIG. 4.-Number of pyroninophilic cells per villus/crypt unit of the proximal jejunum in + /nu or nu/nu mice at various days after oral infection with 100 T. spiralis larvae (mean values ± s.e. mean of groups of 5 animals).

groups was reduced due to a shortening of the villi and a deepening (hyperplasia) of the crypts. Scalloping of the epithelial lining was virtually absent. At Day 7 p.i. the ratio was approximately 2: 1 (Fig. 2). At Day 0 the villus/crypt ratio in both nu/nu and +/nu animals was the same (Fig. 3). After infection the villus/ crypt ratio of both groups showed an initial increase (Day 3 p.i.) and then a decrease (Day 7 p.i.) after which a return to normal values was observed during the rest of the observation period. At Day 0 the mitotic index of the + /nu animals was significantly higher than that of the nu/nu animals. After

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E. J. RUITENBERG, F. LEENSTRA AND A. ELGERSM

infection the mitotic index of the nu/nu groups showed a significant increase from Day 7 to Day 13 p.i. In the +/nu animals no marked changes were seen until Day 10 p.i. At Days 13 and 16 p.i. a significant decrease was observed. At day 0 the number of pyroninophilic cells in the nu/nu animals was significantly lower than that in the +/nu mice (Fig. 4). After infection the number of cells increased significantly in the + /nu mice until Day 10 (peak response), after which a significant decline was observed. In the nu/nu animals an initial decrease of the number of pyroninophilic cells until Day 7 p.i. was followed by an increase at Day 10 (the mean number of cells being under control nu/nu values) and a decrease until the end of the observation period (Day 16 p.i.). DISCUSSION

From the data presented in this paper it may be concluded that changes in the villus/crypt ratio in the T. spiralisinfected animals (both +/nu and nu/nu mice) showed a similar pattern. The pattern in the infected + /nu mice is compatible with the expulsion of the T. spirali8 adult worms at Day 10 (Ruitenberg and Steerenberg, 1974). After expulsion the gut morphology returned to normal. It is interesting to note that the villus/crypt ratio in the infected nu/nu mice showed also an initial decrease, indicating that in the absence of T cells intestinal damage occurred. Surprisingly, however, the villus/crypt ratio of the infected nu/nu mice was the same as in the infected + /nu mice at Days 13 and 16, when worms were still present in the nu/nu animals (Ruitenberg and Steerenberg, 1974). On the other hand, the mitotic indices in the infected nu/nu mice were increased at Days 10, 13 and 16. The higher mitotic index is an indication for an increased turn-over rate of the epithelial cells and an active repair of the integrity of the intestinal epithelial

lining. Pyroninophilic cell production including increase in the number of plasma cells

was only observed in the infected + /nu mice. This is in accordance with the observation that only + /nu mice produced specific antibodies to T. spiralis, indicating T-cell dependence of T. spiralis antigens (Ruitenberg et al., 1977). Based on the results of this experiment the following conclusions may be drawn: First, the non-specific histopathological reactions (changes in villus/crypt ratio or increase of mitotic index) occurred independently of the host immune status, i.e. they represent thymus-independent phenomena. Next, the specific (immunological) reaction (pyroninophilic cell production) is dependent on the immune status of the host. The first conclusion is in contrast to the findings of Ferguson and Jarrett (1975), who suggested that non-specific histopathological gut changes (i.e. villus/crypt ratio) was thymus-dependent. In support of our present observations is the villus atrophy in nu/nu mice observed during a severe infection with the intestinal flagellate Hexamita muris (Berkvens, unpublished) and the marked decrease in villus/crypt ratio in nu/nu mice observed after oral infection with Listeria monocytogenes (Ruitenberg, van Noorle Jansen and Elgersma, unpublished). REFERENCES FERGUSON, A. & JARRETT, E. E. E. (1975) Hypersensitivity Reactions in the Small Intestine. I. Thymus Dependence of Experimental Partial Villous Atrophy. Gut, 16, 114. JARRETT, W. F. H., JARRETT, E. E. E., MILLER, H. R. P. & URQUHART, G. M. (1968) Quantitative Studies on the Mechanism of Self-cure in Nippostrongylus brasiliensis Infections. Proc. 3rd. Int. Congr. Wld. Ass. Adv. Vet. Parasit. In The Reaction of the Host to Parasitism, Ed. E. J. L. Soulsby. Elwert, Marburg/L.: p. 191. REILLY, R. W. & KIRSNER, J. B. (1965) Runt Intestinal Disease. Lab. Invest., 14, 102. RUITENBERG, E. J., ELGERSMA, A., KRIUSINGA, W. & LEENSTRA, F. (1977) Trichinella spiralis infection in Congenitally Athymic (nude) mice. Parasitological, serological and haematological studies with observations on intestinal pathology. Immunology., in the press. RUITENBERG, E. J. & STEERENBERG, P. A. (1974) Intestinal Phase of Trichinella spiralis in Congenitally Athymic (nude) Mice. J. Parasit., 60, 1056. SWANSON, V. L. & THOMASSEN, R. W. (1965) Pathology of the Jejunal Mucosa in Tropical Sprue. Am. J. Path., 46, 511.

Thymus dependence and independence of intestinal pathology in a Trichinella spiralis infection: a study in congenitally athymic (nude) mice.

Br. J. exp. Path. (1977) 58, 311 THYMUS DEPENDENCE AND INDEPENDENCE OF INTESTINAL PATHOLOGY IN A TRICHINELLA SPIRALIS INFECTION: A STUDY IN CONGENITA...
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