THE ANATOMICAL RECORD 208:427-433 (1984) Lymphocyte Proliferation in Neonatally Thymectomized Rats ANNA LUISA GRANATA, C. VECCHI, R. BUDASSI, AND A. CORSI Laboratory of General Pathology, University of Anconq 60100 Anconq Italy ABSTRACT Autoradiography has been used to evaluate lymphocyte proliferation in the neonatally thymectomized rat in comparison with the normal animal. The data obtained show that the proliferative activity of lymphocytes is greatly increased in the thymus-dependent areas 4-6 weeks after thymectomy, whereas it is normal or slightly increased 3 months later. It seems plausible to assume that a thymus factor or chalones in situ produced by specific cells normally regulate the proliferation of thymus-derived cells. The increase of the proliferative activity accounts for the repopulation of the thymus-dependent areas, which are completely replenished in the older animals. Recirculation of the thymus cells is not confined to the thymus-dependent areas. It is generally recognized that the thymus generates lymphocytes which migrate to specific areas in the peripheral lymphoid tissues, particularly in the neonatal life (Parrott et al., 1966; Weissman, 1967; Laissue et al., 1976). The main sites of the thymus cell migration are identical with the areas of lymphocyte depletion appearing in the spleen and lymph nodes after neonatal thymectomy. These areas were delineated in the rat by Goldschneider and McGregor (1973), in basic agreement with observations in the mouse (Parrott et al., 1966). Thymus cells also migrate to the internodular areas of the Peyer's patches (Eikelenboom et al., 1979).The available data do not allow us to define the relevance of cell migration from the thymus or the role of the proliferation of the migrated cells in the development and maintenance of the thymus-dependent pool. It was suggested that the thymus affects the lymphocyte proliferation in the peripheral lymphoid organs by a stimulating factor (Metcalf, 1956, 1964). This possibility was not ruled out by more recent experiments with thymus grafts (Papiernik and Bach, 1979) or with isolated thymus factors (Trainin and Small, 1970; Basch and Goldstein, 1974; Bach et al., 1975). A markedly reduced lymphocytopoiesis in young thymectomized rats was reported by Rieke and Schwarz (1966). Their observations in the lymph and blood do not seem to be entirely congruent with their data in the lymph nodes: the labeling pattern of the lymph and blood lymphocytes as reported by them points to increased proliferative po0 1984 ALAN R. LISS, INC. tential of lymphocytes after thymectomy, whereas the reduced labeling in their radioautographs of sections of lymph nodes is evidence to the contrary. However, they did not quantify the labeling pattern in the thymus-dependent areas. The purpose of this study was to evaluate the proliferative activity of lymphocytes both in the thymus-dependent and in the thymusindependent areas of the peripheral lymphatic organs and in bone marrow after neonatal thymectomy. MATERIALS AND METHODS Animals All experiments were carried out with Wistar albino rats that were bred in our animal colony. Thymectomy Thymectomy was performed by suction according to the method of Miller (19621, mostly from 24 to 36 hours after birth. The time interval never exceeded 38 hours. The efficacy of the procedure was checked in each animal by dissection after killing. Small residues were found only in one of five operated animals, those with residues over 20 mg being rejected. Twenty milligrams was chosen as a safe limit both for theoretical rea- Received June 17,1982; accepted October 3,1983. Address reprint requests to Dr. A.L. Granata, Laboratorio di Patologia Generale, Facolta di Medicina dell'universita, 60100 Ancona, Italy. 428 A.L. GRANATA, C. VECCHI, R. BUDASSI, AND A. CORSI sons (the weight of thymus at 6 weeks of age is over 300 mg) and for experimental data: in our experience a residue of 30 mg a t 6 weeks does not affect appreciably the histologic changes caused in the secondary lymphoid organs by early thymectomy. Labeling Procedure The animals were divided into three groups. Animals in the first group, including thymectomized and control unoperated rats 6 weeks of age, were injected intravenously with a single dose of 1 pCilg body weight of [meth~l-~Hlthymidine 2 Cilmmol (the Radiochemical Center, Amersham, England) and were killed 15 min after the injection. Animals in the second group, including thymectomized and unoperated rats 4-5 weeks of age, were injected intraperitoneally with 3 doses of 1 pCilg body weight of tritiated thymidine every 2 hours and were killed 4 days later. Animals in the third group, including thymectomized and unoperated rats 4 months of age, were treated in the same way as the first group and were killed 15 min after the injection. Histology Smears of blood and lymph from the thoracic duct were made immediately prior to killing and fixed in methanol. Samples of the spleen, mesenteric, and axillary lymph nodes and Peyer’s patches were fixed in a modified Carnoy’s solution (65% ethanol absolute, 30% chloroform, 5% acetic acid) and embedded in paraffin. Femoral bone marrow samples were diluted with homologous serum and fixed in methanol. A morphometric study of Malpighia n bodies was carried out in six normal and five thymectomized animals, 4 months of age, by a point-counting method (Rohr et al., 1976). Counts of the intersection points of a reticulum ocular within the profile of each Malpighian body were done on random sections of 50 bodieslanimal with a magnification of ~ 1 2 5 The . significance of the data was tested by analysis of variance. A u toradiography The tissue sections and the smears were processed for autoradiography by dipping the slides in the Eastman Kodak NTB-2 emulsion. Exposure times of 26 and 23 days were employed for smears and tissues, respectively. The tissue sections were stained with haematoxylin and eosin. The smears of blood, lymph, and marrow cells were stained with haematoxylin, eosin, and Azur B. At least 200 lymphoid cells were counted in each area of the organs and in each sampie of blood, lymph, and bone marrow, and the percentage of labeled cells was determined. Three or more radioautographic grains in the nuclei were assumed to indicate positive labeling by comparison with counts of background grains. The significance of experimental results was determined by the parametric Student’st-test. RESULTS Histology Our findings 4-6 weeks after thymectomy are in accordance with the observations of Parrott et al. (1966)in the mouse and of Goldschneider and McGregor (1973) and Eikelenboom et al. (1979) in the rat. Lymphocytes were sparsely present around the central arteriole of the spleen nodules (Fig. 1). The lymph nodes were smaller than in normal rats of the same age: the cortex was thinner and often depleted of lymphocytes in the mid and deep zone. On the whole, the histologic differences from the normal were not a s constant and obvious in the lymph nodes as in the Malpighian bodies. In older thymectomized animals several observations consistently showed that the morphology of the lymphoid organs progressively approached normality. After 4 months a slight reduction of size was seen only in occasional lymph nodes. The appearance of the Malpighian bodies was almost always normal (Fig. 2); morphometric analysis excluded a significant difference in size of the Malpighian bodies between normal and thymectomized animals [F = 1.625, 10, and 539 degrees of freedom (DF); P > 0.11. Labeling Pattern of the Lymphoid Cells 15 Minutes After the Administration of the Isotope 6 Weeks After Thymectomy A greater percentage of lymphoid cells were labeled in the periarteriolar sheaths of the spleen and in the deep cortex of the mesenteric lymph nodes of thymectomized animals than in controls. The labeled cells were on an average 12.2% in the periarteriolar sheaths of the spleen and 7.4% in the deep cortex of the mesenteric lymph nodes of thymectomized rats. By contrast, the average numbers of labeled cells in controls were 2.6% and 2.2%, respectively (Table 1).The differences were highly significant (P < 0.001). In the thymectomized animals a percentage greater than normal of lymphoid cells were labeled also in the internodular zones of the LYMPHOCYTE PROLIFERATION AFTER THYMECTOMY Fig. 1. A. Spleen of a normal 42-day old rat. The periarteriolar area is densely populated by lymphocytes. B. Spleen of a neonatally thymectomized 42-day old rat. Lymphocytes are sparsely present around the central arteriole. Magnification, x 150. 429 430 A.L. GRANATA, C. VECCHI, R. BUDASSI, AND A. CORSI Fig. 2. A. Spleen of a normal 4-month old rat. Note the large secondary nodule. B. Spleen of a neonatally thymectomized 4-month old rat. Note the large secondary nodule. Magnification, ~ 1 5 0 . 431 LYMPHOCYTE PROLIFERATION AFTER THYMECTOMY TABLE 1. Percentage o f 9 m p h o i d cells labeled 15 m i n after a single dose of [methylHlthymidine in rats 6 weeks o f age Normal Spleen Periarteriolar sheaths Nodules Marginal zone Mesenteric lymph nodes Outer cortex Deep cortex (paracortex) Medullary cords Germinal centers Axillary lymph nodes Outer cortex Deep cortex (paracortex) Medullary cords Peyer’s patches Internodular areas Nodules Germinal centers Bone marrow Thymectomized + 2.6 f 0.7 (7) 2.1 f 0.3 (6) 1.7 f 0.5 (7) 12.2 2.1 (7) 2.1 k 0.6 (6) 1.9 i 0.2 (7) 2.4 f 0.7 (6) 2.2 k 0.4 (6) 7.1 0.4 (6) 12.0 1.9 (6) 3.1 _+ 0.9 (8) 7.4 f 1.0 (8) 9.6 k 1.1(8) 13.9 ? 2.1 (8) 2.8 _+ 0.8 (7) 5.7 f 0.7 (7) 5.6 f 1.9 (6) 2.3 f 0.4 (7) 6.3 k 1.0 (7) 10.6 _+ 1.4 (7) 3.7 f 1.1(6) 8.3 f 3.3 (6) 12.4 k 2.6 (6) 2.4 + 0.5 (4) 8.1 i 1.7 (7) 13.9 f 2.5 (7) 11.4 k 2.0 (7) 2.5 0.6 (4) *+ + Results are the means & SEM of (n) animals. The areas in the spleen and in the lymph nodes were delineated according to Goldschneider and McGregor (1973). Peyer’s patches and in the medullary cords of the axillary lymph nodes; the differences from the normal values were near to significant (P < 0.1) (Table 1). Few lymphoid cells were labeled in bone marrow (2.5%) and in blood (0.5% or less), and no change was seen after thymectomy. Labeling Pattern of Lymphoid Cells 4 Days After the Administration of the Isotope 4-5 Weeks After Thymectomy The results are summarized in Table 2. The labeling pattern was altered throughout the white pulp of the spleen: the percentage of labeled lymphoid cells was greater in the thymectomized animals than in controls, the difference being significant (P < 0.01) in the area immediately surrounding the central arteriole and in the marginal zone. The mean values were 32.4% and 12.9%, respectively, after thymectomy, whereas only 7.5% and 4.2%, respectively, were labeled in control animals. A significant increase of labeled cells was found in the medullary cords of the axillary lymph nodes after thymectomy (P < 0.05); the mean percentage of labeled cells was 39.6 in the experimental animals and 23.0 in controls. A highly significant difference (P < 0.001 and P < 0.01, respectively) was seen in blood and lymph. The percentage of labeled lymphocytes in blood was on a n average 42.5 after thymectomy and 23.7 in controls. The mean values in lymph were 22.1 and 11.8, respectively. No appreciable difference was seen in the labeling pattern of lymphoid cells in the bone marrow (39.5% after thymectomy and 42.0% in controls). Labeling Pattern of Lymphoid Cells 15 Minutes After the Administration of the Isotope 4 Months After Thymectomy The labeling pattern of lymphoid cells was investigated in the spleen and the mesenteric lymph nodes. The results are shown in Table 3. The labeled cells were on a n average 2.8% in the periarteriolar sheaths of the spleen and 3.0% in the deep cortex of the mesenteric lymph nodes of thymectomized animals. The average numbers in the control animals were 1.7% and 1.5%, respectively. The difference was significant (P < 0.01) only in the deep cortex of the mesenteric lymph nodes. The percentage of labeled cells 4 months after thymectomy was greatly reduced in the spleen and in the deep cortex of the lymph nodes, if compared with the data in younger animals 6 weeks after thymectomy: the difference was significant. The reduction in the percentage of labeled cells was 432 A.L. GRANATA, C. VECCHI, R. BUDASSI, AND A. CORSI TABLE 2. Percentage of lymphoid cells labeled 4 days after administration of [methyl3H]thymidine in rats 4-5 weeks of age Spleen Periarteriolar sheaths Nodules Marginal zone Axillary lymph nodes Outer cortex Deep cortex (paracortex) Medullary cords Germinal centers Peyer’s patches Internodular areas Nodules Germinal centers Bone marrow Blood Lymph Normal Thymectomized 7.5 f 2.3 (6) 10.2 f 2.2 (6) 4.2 f 1.0 (6) 32.4 f 5.2 (6) 18.6 f 3.7 (6) 12.9 f 1.8 (6) 11.1k 0.6 (6) 14.6 ? 2.0 (6) 16.4 f 3.4 (6) 39.6 f 6.1 (6) 19.0 f 2.9 (3) 12.6 k 0.8 (6) 23.0 k 3.3 (6) 39.0 f 6.5 (5) 17.6 16.3 31.6 42.0 23.7 11.8 5 3.0 (5) f 1.9 (5) f 3.4 (5) k 3.7 (5) f 3.3 (6) f 1.9 (5) 19.2 17.6 25.6 39.5 42.5 22.1 f 1.1(5) f 2.7 (5) f 6.8 (6) f 5.4 (5) 5 5.3 (7) & 1.7 (5) Results are the means SEM of (n) animals. The areas in the spleen and in the lymph nodes were delineated according to Goldschneider and McGregor (1973). TABLE 3. Percentage lymphoid cells labeled 15 m i n after a single dose of [methyl3H]thymidine in rats 4 months of age of Spleen Periarteriolar sheaths Nodules Marginal zone Mesenteric lymph nodes Outer cortex Deep cortex (paracortex) Medullary cords Germinal centers Normal Thymectomized 1.7 f 0.2 (4) 0.6 0.1 (4) 0.5 f 0.1 (4) 2.8 i 0.4 (5) 0.7 f 0.1 (5) 0.5 f 0.1 (5) 0.9 k 0.2 (4) 1.5 + 0.1 (4) 1.5 f 0.7 (5) 3.0 0.3 (5) 5.9 k 2.6 (5) 9.8 f 1.0 (5) * 5.6 f 2.0 (4) 8.6 f 1.4 (4) + Resuts are the means 5 SEM of (n) animals The areas in the spleen and in the lymph nodes were delineated accordmg to Goldschneider and McGregor (1973). small in normal older animals: the difference in comparison with normal younger animals was not significant. though the actual number of dividing cells may be smaller than normal because the total thymus-dependent pool is smaller, the increased proliferative activity soon after DISCUSSION thymectomy affords a satisfactory explanaThe labeling pattern demonstrates that the tion of why the thymus-dependent areas are percentage of dividing cells is generally in- replenished a few months later. Our results show that a thymus factor is creased in the thymus-dependent areas of the rat 4-6 weeks after neonatal thymectomy: 4 not required for the proliferation of the thymonths after thymectomy the labeling pat- mus-derived cells. By contrast, our results tern approaches normality and the thymus- are compatible with the view that a circulatdependent areas are replenished. It appears ing thymus factor normally inhibits the prothat the proliferative activity of the early- liferation of specific cells. In fact, a thymic migrating thymus cells is markedly in- inhibitor of lymphocyte proliferation was isocreased 4-6 weeks after thymectomy. Al- lated by Rijke and Balliew (1979). It also LYMPHOCYTE PROLIFERATION AFTER THYMECTOMY seems plausible that in situ-produced chalones play a role in the regulation of the thymus-dependent pool. A soluble fraction capable of inhibiting proliferation of lymphocytes was obtained by Moorhead et al. (1969). The repopulation of the thymus-dependent areas was reported by Parrott et al. (1966) in occasional thymectomized mice surviving 67 weeks: the authors had no satisfactory explanation for their finding, which seems to have been ignored by the other research workers in this field. One might speculate that a difference in the regulation of development and maintenance of the lymphatic pools may account for the much greater proneness of mice to runt disease after thymectomy in comparison with rats. It is worth noting that in the thymectomized animals 4-5 weeks of age, examined a t 4-day intervals after administration of tritiated thymidine, the difference in the labeling pattern is not confined to the thymusdependent areas. This is most likely the consequence of further cell migration and recirculation. ACKNOWLEDGMENTS The authors are indebted to Professor G. Doria and Professor Z. Ovary for their helpful comments on this manuscript. The technical assistance of Mr. V. 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