ARTICLE |
Correspondence to: Jürgen Westermann, Zentrum Anatomie 4120, Medizinische Hochschule Hannover, 30623 Hannover, Germany.
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Summary |
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To study the lifespan of immune cell populations in the LEW rat, 5-bromo-2-deoxyuridine (BrdU) was administered in the drinking water. After 12 weeks, the epithelium of gut and skin was completely BrdU+. In contrast, thymus, bone marrow, and germinal centers of Peyer's patches contained only a few BrdU+ cells, although most should have been labeled during this time. The lack of labeling was due neither to obvious toxic effects of BrdU on these organs nor to insufficient detection of incorporated BrdU. Analysis of the kinetic pattern of the appearance of BrdU+ cells in bone marrow, blood, spleen, and lymph nodes over 12 weeks revealed that the dosage of BrdU initially was high enough to label the proliferating cells in the bone marrow, but then became too low, although the BrdU uptake of the rats was similar over the entire time. This indicates that in the LEW rat the metabolism of orally applied BrdU changes over time, leading to a reduction in the amount of BrdU available for incorporation into the DNA below a level necessary for labeling all proliferating cells. This effect appears to be species- and strain-dependent, and should be considered when the BrdU technique is used. (J Histochem Cytochem 45:393-401, 1997)
Key Words: proliferation, oral application, BrdU, long-term labeling, LEW rat, Balb/c mouse, thymus, bone marrow, blood, spleen, lymph nodes
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Introduction |
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The lifespan of B- and T-lymphocytes and natural killer cells is an important factor in regulating the size of the respective population, and thereby determines to a great extent the capacity of the immune system. One possibility for studying the lifespan of cell populations is to label all newly formed cells over a defined period of time with BrdU, a DNA precursor (
Therefore, in the present study not only the proliferation of B- and T-lymphocytes and natural killer cells in the LEW rat was determined but also great care was taken to detect possible adverse effects of long-term BrdU application in this species. LEW rats were given an often-used BrdU dosage in the drinking water over 12 weeks (1 mg/ml) (e.g.,
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Materials and Methods |
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Animals and BrdU Application
Adult male LEW rats (n = 25) and Balb/c mice (n = 14) were kept under specific pathogen-free conditions until the beginning of the experiments. The animals had free access to food and water. Rats and mice were continuously administered BrdU (5-bromo-2-desoxyuridine; Sigma, Munich, Germany) for various times up to 12 weeks. BrdU (150 mg) was dissolved in 125 ml water and 25 ml orange juice (previous studies showed that the amount of BrdU water imbibed was reduced when the orange juice was lacking). The BrdU concentration was 1 mg/ml water according to established protocols (e.g.
Cell Preparations and Histological Sections
Rats were exsanguinated under ether anesthesia after 3, 17, 24, 38, 52, 59, 70, or 84 days (12 weeks) of BrdU administration. Balb/c mice were investigated after 7, 17, 24, or 70 days of BrdU administration. The number of leukocytes and the percentage of lymphocytes and granulocytes in the arterial blood were determined. The organ weights of the thymus, spleen, mesenteric lymph nodes, axillary lymph nodes, liver, right kidney, and right testicle were recorded and the organs were prepared for histology and cytology. Tissue samples of the thymus, skin, small intestine, and Peyer's patches were quickly frozen in liquid nitrogen and cryosections 5 µm thick were made. After air-drying the slides were wrapped in aluminum foil and stored at -20C. Thymus, spleen, and mesenteric lymph nodes were minced and washed with RPMI 1640 (Seromed; Berlin, Germany) at 400 x g for 10 min. The red blood cells were removed by osmotic shock. Afterwards, the cells were centrifuged through 2 ml of fetal calf serum (Seromed) at 400 x g for 10 min and resuspended in RPMI 1640 with 0.5% bovine serum albumin (BSA; Serva, Heidelberg, Germany). To obtain the bone marrow cells, the femur was flushed with RPMI 1640 containing 0.5% BSA and the cells were treated as described above. The blood was diluted 1:4 with RPMI 1640 containing 0.5% BSA. After centrifugation in Ficoll Isopaque (Pharmacia; Uppsala, Sweden) (= 1089 g/cm3 at 400 x g for 30 min) the interface layer was harvested and washed (800 x g for 10 min). Then cells were treated as described above. Cytocentrifuge preparations were made (Shandon, Frankfurt, Germany; 150 x g, 8 min). To prevent clotting, 0. 1 x 106 cells were diluted with 100 µl 0.9% NaCl (containing 2.7 mM Na-EDTA, 5% BSA, pH 7.2). The slides were air-dried overnight, wrapped in aluminum foil, and stored at -20C.
Characterization of the Phenotype and Detection of Incorporated BrdU by Immunostaining
The cell surface was stained by the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique (
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Evaluation
The percentages of the various phenotypes, the BrdU+ cells, and the double-positive cells (phenotype+ and BrdU+) were determined by counting at least 100 positive cells. Means and standard deviations were calculated.
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Results |
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Long-term BrdU Labeling Did Not Cause Obvious Toxic Effects
Over the 12-week period, the daily BrdU uptake was in the range of 20 mg, leading to a total amount of orally administered BrdU of 1439 ± 103 mg (n = 10). Obvious toxic effects of the incorporated BrdU were not seen. The animals behaved normally and showed no clinical signs of illness. The histological appearance of thymus and Peyer's patches was not altered compared to control animals (Figure 1), nor was the functional capacity of the bone marrow, as indicated by a normal number of granulocytes in the blood (Table 2). In addition, the body weight, the organ weights, the blood lymphocyte numbers (Table 2), and the cellular composition of bone marrow, blood, spleen, and mesenteric lymph nodes were not altered by BrdU application compared to those of control animals (Table 3).
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The Epithelium of Gut and Skin Was Completely Labeled by BrdU but only a Few Cells in Thymus and Bone Marrow Were BrdU+
As expected, sections of the gut and the skin of the rats showed that all epithelial cells had incorporated BrdU after 12 weeks of administration (Figure 2). On cytological preparations of blood, spleen, and mesenteric lymph nodes, B- and T-lymphocytes and natural killer cells could clearly be identified and incorporated BrdU revealed (Figure 3). After 12 weeks of BrdU administration, 30.3 ± 0.7% (n = 5), 28.9 ± 0.9% (n = 5), and 20.2 ± 0.6% (n = 5) BrdU+ cells were found in blood, spleen, and mesenteric lymph nodes, respectively. The percentage of BrdU+ cells among the different subsets varied considerably, being 15% for T-lymphocytes in the mesenteric lymph nodes and 75% for natural killer cells in the blood (Figure 4). B- and T-lymphocyte subpopulations (IgM+ and IgD+ cells, and CD4+ and CD8+ cells, respectively) showed a much smaller heterogeneity (15-25% of the cells of the respective subset being BrdU+). However, surprisingly few cells in the rat thymus (0.7 ± 0.4%, n = 5) and bone marrow (12.8 ± 1.0%, n = 5) were BrdU+ after 12 weeks. In the thymus, BrdU+ cells were located in the medulla (Figure 1). Cytological preparations of the rat bone marrow showed that after 12 weeks of BrdU administration 97.8 ± 0.4% (n = 5) of the monocytic cell lineage (ED1+) and 97.6 ± 2.4% (n = 5) of the neutrophilic granulocytes (RP1+) had not incorporated BrdU. In addition, in germinal centers of Peyer's patches, only a few lymphocytes were BrdU+, whereas the corona contained many BrdU+ cells (Figure 1). To check whether the low number of BrdU+ cells in thymus, bone marrow, and germinal centers of Peyer's patches in the present study might be due to insufficient BrdU detection by immunostaining, our technique was applied to Balb/c mice given BrdU (1 mg/ml in the drinking water) and was compared in the same experiment to similarly treated rats. It was obvious that most cells in the thymus and bone marrow of Balb/c mice were BrdU+ after 2 and 10 weeks of labeling (Figure 5). After 3 weeks, 90 ± 2% (n = 4) BrdU+ cells were found in the mouse thymus, as determined on cytospin preparations. After 2, 3, or 10 weeks of BrdU application 87 ± 5% (n = 2), 92 ± 2% (n = 4), and 94 ± 2% (n = 4) had incorporated BrdU into mouse bone marrow. No labeling at all could be detected when BrdU was omitted from the drinking water (Figure 5). In LEW rats, labeled cells were detected in the thymic medulla but not in the cortex (both after 2 and 10 weeks of labeling) (Figure 5). In the bone marrow of LEW rats many BrdU+ cells were found after 2 weeks of labeling (61 ± 9%) but only very few after 10 weeks (20 ± 15%; Figure 5). As in mice, omitting BrdU from the drinking water led to no BrdU staining at all (Figure 5).
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Time-course Analysis Showed that the Number of BrdU+ Cells First Increased but then Decreased, Although the BrdU Uptake Was Constant
When the numbers of BrdU+ cells were studied in LEW rats at several time points in various organs during a 12-week labeling period, it became obvious that the number of BrdU+ cells did not increase constantly (Figure 6). The most dramatic changes were observed in the bone marrow (Figure 6B). After 2 weeks of labeling, about 70% of all bone marrow cells were BrdU+. Then the number declined to reach a level of about 10% after 12 weeks of BrdU labeling. An increase and a subsequent decrease in the numbers of BrdU+ cells were also seen for blood, spleen, and lymph nodes, although less pronounced (Figure 6C and Figure 6D). When the staining intensity of the gut and skin epithelium (which is proportional to the amount of incorporated BrdU) was investigated over time, similar changes were observed (data not shown). Surprisingly, during the entire time the BrdU+ uptake of the rats was constant (Figure 6A).
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Discussion |
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BrdU is commonly used to label proliferating cells during S-phase of the cell cycle (
Because the denaturation protocol is critical in revealing incorporated BrdU (
The few BrdU+ cells in thymus, bone marrow, and the germinal centers of Peyer's patches are also not due to gross toxic effects of BrdU. Histological examination showed a normal architecture of thymus and Peyer's patches (Figure 1), and cytological examination a normal cellular composition of the bone marrow (Table 3). In addition, the numbers of granulocytes in the blood, which are predominantly produced in the bone marrow, were not affected by BrdU administration (Table 2). The unchanged body and organ weights, and the normal cellular composition of blood, spleen, and lymph nodes, also argue against toxic effects of BrdU in our experiments (Table 2). Even toxic effects of BrdU resulting in the death of only a small number of cells should be picked up by the parameters studied because, owing to the long BrdU administration period, such effects would accumulate with time and most likely would result in detectable damage. However, by largely unknown mechanisms (
Because our technique is as sensitive as techniques described by other groups (
One reason that such a change in the metabolism of BrdU has not been described thus far might be the fact that it depends on both the species and the animal strain. When, instead of LEW rats e.g., Balb/c mice (
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Acknowledgments |
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Supported by the Deutsche Forschungsgemeinschaft (Je 201/1-1 and We 1175/4-1).
The generous donations of antibody 3.2.3 by Dr R. Herberman, (Pittsburgh Cancer Institute) and of antibody RP1 by Dr F. Sendo (Yamagata University) are gratefully acknowledged. The technical assistance of Ms P. Schwarz, Ms A. Herden, and Ms. S. Lopez-Kostka, the help by Ms D. Stelte in preparing the figures, and the correction of the English by Ms S.Fryk are deeply appreciated.
Received for publication September 10, 1996; accepted October 14, 1996.
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