SMALL INTESTINAL EPITHELIAL CELL KINETICS AND PROTOZOAL INFECTION IN MICE

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1 /78/ ~02.00/0 GABTROENTEF.OLOGY Copyright Q 1979 by the American Gastroenterological Association Vol. 74, No. 3 Printed in USA. SMALL INTESTINAL EPITHELIAL CELL KINETICS AND PROTOZOAL INFECTION IN MICE T. T. MACDONALD, PH.D., AND ANNE FERGUSON, PH.D., F.R.C.P., M.R.C.PATH. Department of Bacteriology and Immunology, University of Glasgow, and Gastro-Zntestinal Unit, University of Edinburgh and Western General Hospital, Edinburgh, Scotland The effects of chronic protozoa1 infection on small intestinal architecture have been examined in mice, infected with Giurdia murk and Hexamitu muris. Techniques used were conventional histology, quantitation of intraepithelial lymphocytes, microdissection and measurement of individual villi and crypts, and epithelial cell kinetic studies. The histology of small intestine from infected mice appeared normal apart from the intraepithelial lymphocyte numbers. Mean intraepithelial lymphocyte counts in two groups of uninfected mice were 11.6 and 13.6 per 100 epithelial cells, and in two groups of infected mice were 17.6 and Dynamic studies showed that protozoa1 infection doubled the cell production per crypt per hour from mean values of 6.2, 7.3, and 8.2 in three groups of uninfected animals, to 11.8, 13.4, and 17.1 in groups of chronically infected mice. Cell production per villus was also influenced by protozoa1 infection, with values of 93, 99, and 101 cells per hr in groups of uninfected animals whereas in infected mice the values were 155, 162, and 180 cells per hr. Although there was no reduction in villus height in the infected animals, radioautography using 13Hlthymidine confirmed that the enterocytes moved more rapidly up the sides of the villi than was the case for uninfected mice. Giardia and Hexamita are lumen-dwelling flagellates found in the small intestine of man and animals. Human infection with Giardia is often asymptomatic but heavy infections can produce villous atrophy and a malabsorption syndrome. 1-4 Giardia and Hexamita may also cause damage to the small intestine of mice, and protozoa1 infection is thought to contribute to postweaning mortality in both normal and congenitally athymic mice.5* 6 In the course of a research program on the nature of small intestinal lymphocytes, we found that intraepithelial (IE) lymphocyte counts were high in one of the three strains of mice maintained in our animal house. Further investigation showed that Giardia and Hexamita infections were endemic in this strain, but that mice of the other two strains were free from infection. This provided an opportunity to investigate the influence of chronic protozoa1 infection on intestinal architecture and epithelial cell kinetics in mice, and also to examine the interrelationships between such infection, IE lymphocyte counts, and changes in epithelial cell kinetics. Materials and Methods Animals. The animals used in this study were adult mice of the CBA, BALBlc, and Sha-Sha strains, maintained in the Received November 15, Accepted September 21, Address requests for reprints to: Dr. Anne Ferguson, Gastro- Intestinal Unit, Western General Hospital, Edinburgh EH4 2XU Scotland. This study was supported by grants from the National Fund for Research into Crippling Diseases and the Camilla Samuel Fund. The authors wish to thank Mr. H. Cairns who processed the histology and Mrs. H. Collin who prepared the radioautographs. 496 Department of Bacteriology and Immunology, University of Glasgow. Details of the various groups of animals studied are summarized in table 1. With the exception of group 4, all of the mice were aged between 3 and 6 months. Group 4 mice were aged 6 weeks. At the onset of the study, in 1974, CBA and BAB/c mice (groups 1 and 2) were free from protozoa1 infection but the Sha-Sha breeding stock were infected with Giardia and Hexamita and these parasites were found in the intestines of all Sha-Sha mice from the time of weaning (group 3). Artificial infection of mice of the CBA strain (group 4) was carried out by fostering lo-day-old CBA babies to infected Sha-Sha mothers. Examination of the jejunal contents of these mice 4 weeks later showed that they harbored both Giardia and Hexamita. Nine months after the initial observations had been made, the animal unit was redecorated. For 2 months there was a temporary breakdown in the standard of animal handling and maintenance necessary to prevent cross infection between the strains, and some but not all of the mice of the BALB/c strain were found to have become infected with Giardia. Group 5 was comprised of BALB/c mice remaining uninfected, and group 6 of BALB/c mice accidentally infected at this time. Histology of small intestine. Mice were killed by cervical dislocation, and 0.5 to l-cm long pieces of proximal jejunum were removed and immediately fixed in formol saline. These were paraffin embedded, and sections 3.5-pm thick were stained with hematoxylin and eosin, Giemsa, and methyl green pyronin. Counts of ZE lymphocytes. An assessment of the degree of lymphocyte infiltration of the epithelium was made by carrying out differential counts of epithelial cells and IE lymphocytes by a method previously described.? A total of 500 villus epithelial cells was counted and the lymphocyte numbers were expressed per 100 epithelial cells. These IE lymphocyte counts were carried out in mice of groups 1, 3, 5, and 6, and

2 March 1978 INTESTINAL ARCHITECTURE AND PROTOZOAL INFECTION 497 TABLE 1. Numbers of mice used to study the effect ofprotozoa Group infection on intestinal epithelial ckll kinetics Strain of IllOUBC? Date Uninfected 1 CBA BALBIC Sha-Sha CBA P 5 BALBic BALBlc Y a Deliberately b Accidentally infected. infected. the numbers of animals examined are indicated in table 2. Radioautography. Six uninfected mice (group 1) and 6 infected mice (group 3) were injected intraperitoneally with 25 &i [3H]thymidine. Twenty-four hours later the animals were killed and a piece of proximal jejunum was fixed in form01 saline. The tissue was embedded in wax and 3.5pm sections were cut and mounted. The sections were coated by dipping them in photographic K5), exposed for 2 to 4 weeks, and then developed, fixed and stained with methyl green pyronin. The position of the leading labeled cells was counted in a minimum of 10 villi per section. Measurements of epithelial cell kinetics. The sizes and relative numbers of crypts and villi were measured after microdissection of fragments of the small intestine as previously described. 0 In addition, the rate of cell production in the crypts of Lieberkuhn was determined in groups of animals, by using colchicine to block mitoses in metaphase. Groups of mice were injected intraperitoneally with colchitine (British Drug House, Poole, England), 5 mg per kg body weight. At intervals from 1 to 3 hr later, the mice were killed by cervical dislocation, a l-cm piece of proximal jejunum was dissected out and fixed in a 75% absolute alcohol-25% acetic acid mixture. The tissue was then stained in bulk by the Feulgen reaction. Measurements of crypts and villi. With a dissecting microscope and by using fine forceps and a cataract knife, single villi with associated crypts were cut free from the edge of the specimen, placed on a slide in a drop of 45% acetic acid, and a cover slip was applied gently. By using an eyepiece graticule in a light microscope the lengths of 10 villi and 10 crypts per specimen were measured. The preparation was gently squashed by light pressure on the cover slip. This liberates the crypts from the lamina propria matrix and flattens them out. The accumulated number of metaphases was then counted in each of 10 crypts per specimen, and the mean value obtained. Number of crypts per uillus. Specimens of small intestine were Feulgen-stained and then processed in the following manner: O 5mm* pieces were placed in 45% acetic acid for 24 hr at room temperature. The tissue destained overnight and was then placed serosa upward on a slide in 45% acetic acid, covered with a cover slip, and examined with a microscope. By using an eyepiece grid, the number of villi and crypts could be counted in the same piece of gut by focusing up and down. Six counts of the number of crypts per villus were made for each piece of tissue and the mean value obtained. Cell production per villus per hour. This was calculated as the product of the number of crypts per villus and the rate of metaphase accumulation per crypt per hour. Statistics. All results are expressed as mean * 1 standard error (SE). Comparison of villus height, crypt depth, and the number of crypts per villus between groups was by Student s t-test. Straight lines were drawn by the method of least squares. Differences in the rate of cell production per crypt per hour (the gradient of the regression lines) were studied by covariance analysis. Results Histology of small intestine in uninfected and in Giardia- and Hexamita-infected mice. Conventional histological examination revealed no gross abnormalities in the intestines of mice of any of the groups studied. Villi were long and slender and the crypts were relatively short. Examples of histology are illustrated in figures 1 and 2. Counts of IE lymphocytes were carried out in the histological specimens of groups 1 and 5 (uninfected and groups 3 and 6 (infect&. These showed that the infected BALB/c and Sha-Sha mice had significantly higher IE lymphocyte counts than did the uninfected mice (P < 0.05, table 2). Rates of cell production in the crypts of Lieberkuhn. When using stathmokinetic techniques to measure cell proliferation rates it is necessary to show that metaphase accumulation is linear in the hours after injection of the metaphase-arresting agent (in this case colchitine). For the technique to be valid the birth rate of enterocytes must remain the same throughout the period of the experiment. Figure 3 illustrates the number of accumulated metaphases per crypt at each time interval after colchicine injection in the different groups of animals used for these experiments. An increase in the number of metaphases with time was observed in all of the groups, and this metaphase accoumulation was linear. Thus, for each experimental group the rate of cell production per crypt per hour was calculated as the gradient of the least mean squares line drawn, using the results of metaphase accumulation with time. The cell production per crypt per hour was similar in the uninfected mice of groups 1, 2, and 5 (7.3, 8.2, and 6.2) and in the infected groups 3, 4 and 6, the values were significantly higher at 13.4, 17.1, and 11.8 (P < 0.05 for groups 1 versus 3, 2 versus 3, 1 versus 4, and 5 versus 6). Intestinal architecture and epithelial cell kinetics. Direct measurements of villi and crypts in the microdissected specimens from each group of mice showed no significant differences in villus height among the groups. However mean values for the crypt depth of the infected mice of groups 3, 4, and 6 were 152, 202, TABLE 2. Intraepithelial Experimental.wUP (IE) lymphocyte counts in small intestine No. of IE lymphoc speci- 100 epithell,p cells mens examined MIX&l-l SE 1. CBA , (uninfected) 3. Sha-Sha J (infected) 5. BALB/c (uninfected) 6. BALBlc (infected) Significance of difference between groups P < 0.05 P < 0.05

3 498 MACDONALD AND FERGUSON Vol. 74, No. 3 FIG. 1. Histology of jejunum from an uninfected BALB/c mouse (H & E, x 90). FIG. 2. Histology of jejunum from a BALB/c mouse infected with Giardia and Hexamita (H & E, x 90). Expwmental I 2 3 L Grour! %a-shd CBA* FIG. 3. Accumulation of blocked metaphases in mice of groups 1 to 6 at various times after colchicine injection. Straight lines have been drawn by the method of least squares and the number of points on each line indicates the times at which groups of mice were killed after colchicine injection (at least 4 mice per group). #s 12.3ro.a I2,I t L O.9 9.1t to.a 11.8t1.7 Number of Crypts per Villus FIG. 4. Diagramatic representation of epithelial cell kinetics in the six groups of animals studies. The arrow leaving the crypt represents the rate of cell production per crypt per hour and the or-row leaving the villus represents the rate of cell production per villus per hour (calculated) which, in the steady state, will equal the rate of cell loss per villus per hour. Asterisks indicate groups of animals chronically infected with Giardia and Hexamita.

4 March 1978 INTESTINAL ARCHITECTURE AND PROTOZOAL INFECTION 499 and 150 km, considerably longer than for the uninfected mice of groups 1, 2, and 5, values for which were 119, 123, and 125 pm. Formal statistical analysis showed that the Sha-Sha mice, group 3, had significantly longer crypts than uninfected mice of the other two strains, groups 1 and 2 (P < 0.001). Similarly, comparisons between groups 1 and 4, 2 and 6, showed that in each case, crypts of infected animals were significantly longer than those of uninfected animals (P < 0.05). Because the number of crypts supplying cells for each villus was similar in each of the groups of the mice examined, the calculated cell production per villus per hour was also increased in the infected groups, being 93, 99, and 101 in groups 1, 2, and 5, and 155, 181, and 162 in the infected groups 3,4, and 6. Thus, for mice of both of the previously uninfected strains, CBA and BALB/c, protozoa1 infection had increased crypt cell production and had altered epithelial cell kinetics to the levels found previously only in the chronically infected mice of the Sha-Sha strain. ViZZus enterocyte transit rate. Because the results described above indicated that protozoa1 infection increased cell production per villus per hour without change in villus shape, this implies that enterocytes move more rapidly up the sides of the villi in infected animals. This fact was confirmed by using radioautography to identify the position of cells, previously labeled on the crypts, at the sides of the villi. Twenty-four hours after injection of 13Hlthymidine into uninfected CBA mice (group 11, the leading labeled cell position up the villus had a mean value of 23.8, standard error 3.0. In the infected Sha-Sha mice (group 3) the mean value for the leading cell was 44.3, with standard error 3.9 (P < 0.005). This experiment confirms that a greatly increased villus transit rate is produced by a protozoa1 infection, Discussion The effects of chronic protozoa1 infection on the small intestinal architecture of mice are virtually undetectable by conventional histological examination. However, by using cytokinetic techniques, we have demonstrated that infection with Giardia and Hexamita will double the rate of cell production in the crypts of Lieberkuhn of the small intestine. Increased cell production in the crypts was not accompanied by significant changes in the shapes or sizes of the villi, but radioautographic studies showed that the rate at which enterocytes moved on to and up the sides of the villi was increased in infected animals. Thus, in this animal model, villi of normal length are clothed by relatively immature enterocytes. The mechanism by which Giardia and Hexamita produce these changes in epithelial cell kinetics has not been defined. The parasites may directly damage enterocytes, and there is some electron microscopic evidence to support this concept. Alternatively, it is possible that the local immune response is the cause of crypt hyperplasia. l2 We examined the changes in epithelial cell kinetics produced by the cell-mediated immune reactions of allograft rejection and graft versus host disease, and showed that early in the evolution of these reactions there is a period when there is crypt hyperplasia with increased cell turnover rate but without obvious villous atrophy. lo Cytokinetic changes during this phase 1 of a local cell-mediated immune reaction were virtually identical to those observed in the present experiments in protozoal-infected rnice. Oz l2 During intestinal infection with a helminthic parasite, thymus-dependent immune response, rather than the parasites themselves, causes changes in mucosal architecture.13 In addition, the raised counts of IE lymphocytes, found in association with Giardia infection in man 4, 14, j as well as in these laboratory animals, provides some evidence that lymphocytes are involved in local immune reactions to these protozoa1 parasites. Strain differences in any easily measurable item in laboratory animals are usually attributed to genetic factors. This could also have been the explanation for the interstrain differences in small bowel cytokinetics which we detected in the first series of experiments reported above. However, because cell proliferation in the crypts increased by 134 and 87%, respectively, when animals of the two previously uninfected strains CBA and BALB/c were infected with protozoa1 parasites, it is clear that the original findings in mice of the Sha- Sha strain were not attributable to genetic factors but were merely a result of the parasite infection. The animals used for these experiments have been chronically infected with Giardia and Hexamita from the neonatal period. The effects of this type of infection probably differ from those of controlled Giardia infection in adult animals. In this latter model, shortening of the villi has been reported, 6 but the infection is selflimited and a self-cure mechanism appears to operate. In contrast, it seems that when mice are infected shortly after birth there is no self-cure mechanism, indicating some impairment of the immune response to the parasites. This appears to be an important field for further study REFERENCES Ament ME, Ochs HD, David SD Structure and function of the gastro-intestinal tract in primary immunodeficiency syndromes: a study of 39 patients. Medicine (Baltimore1 52: , 1973 Petersen H: Giardiasis (Lambliasis). Stand J Gastroenterol 7(suppl 14):1-44, 1972 Alp MH, Hislop IG: The effect of Giordia lamb& infestation on the gastrointestinal tract. Australas Ann Med 18: , 1969 Hoskins LC, Winawer SJ, Broitman SA, et al: Clinical giardiasis and intestinal malabsorption. Gastroenterology 52: , 1967 Boorman GA, Van Hooft JIM, Van der Waaij D, et al: Synergistic role of intestinal flagellates and normal intestinal bacteria in a post-weaning mortality of mice. Lab Anim Sci 23: , 1973 Boorman GA, Lina PHC, Zurder C, et al: Hexamita and Giordia as a cause of mortality in congenitally thymusless (nude) mice. Clin Exp Immunol 15: , 1973 Ferguson A, Murray D: Quantitation of intraepithelial lymphocytes in human jejunum. Gut , 1971 Clarke RM: Mucosal architecture and epithelial cell production rate in the small intestine of the albino rat. J Anat 107:X9-529, 1970 Clarke RM: A comparison of metaphase arresting agents and tritiated thymidine autoradiography in measurement of the& rate of entry of cells into mitosis in the crypts of the Lieberkuhn

5 500 MACDONALD AND FERGUSON Vol. 74,No of the rat. Cell Tissue Kinet 4: , 1971 MacDonald TI, Ferguson A: Hypersensitivity reactions in the small intestine. 3. The effects of allograft rejection and of graftversus-host disease on epithelial cell kinetics. Cell Tissue Kinet 10: ,1977 Tandon BN, Puri BK, Gandhi PC, et al: Mucosal surface injury 15. of jejunal mucosa in patients with Giardiasis: an electron microscopic study. Indian J Med Res 62: ,1974 Ferguson A, MacDonald IT: Effects of local delayed hypersensi- 16. tivity on the small intestine. Ciba Found Symp 46: , 1977 Ferguson A, Jarrett EEE: Hypersensitivity reactions in the 14. small intestine. I. Thymus dependence of experimental partial villous atrophy. Gut 16: , 1975 Ferguson A, McClure JP, Townley RRW: Intraepithelial lymphocyte counts in small intestinal biopsies from children with diarrhoea. Acta Paediatr Stand , 1976 Mavromichalis MJ, Brueton MJ, McNeish AS, et al: Evaluation of the intraepithelial lymphocyte count in the jejunum in childhood enteropathies. Gut 17: , 1976 Roberts-Thomson IC, Stevens DP, Mahmoud AAF, et al: Giardiasis in the mouse: an animal model. Gastroenterology 71:57-61, 1971 :

Increased mucosal damage during parasite infection

Gut, 1980, 21, 37-43 Increased mucosal damage during parasite infection in mice fed an elemental diet ANNE FERGUSON, R F A LOGAN, AND T T MACDONALD' From the Gastro-Intestinal Unit, University oj Edinburgh,