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Influence of age, castration, and testosterone on T cell subsets in healthy and leukemia grafted mice
Biology of the Cell 95 (2003) 9–16 www.elsevier.com/locate/bicell
Influence of age, castration, and testosterone on T cell subsets in healthy and leukemia grafted mice Souad Aboudkhil a,b,c, Abdelhamid Zaîd b, Laurent Henry c, Jean-Paul Bureau c,* a
Ufr environnement et santé, Department of Biology, Faculty of Science and Technique, University Hassan II, Quartier Yasmina, BP 146, Mohammedia, Morocco b Ufr environnement et santé, Department of Biology, Faculty of Science, University Moulay Ismaîl, BP 4010, Beni M’hamed, 50 006 Meknes, Morocco c Laboratory of Cell Biology and Molecular Cytogenetics, Faculty of Medicine, Montpellier-Nîmes, Avenue Kennedy, 30900 Nîmes, France Received 2 May 2002; accepted 12 December 2002
Abstract The distribution of T cell subsets in pubertal (2 months) and post-pubertal (10 months) mice showed a significant decrease in the percentage of CD4+ splenocytes and peripheral blood lymphocytes (PBL) with age, unlike the percentage of CD8+ cells in PBL, which remained unchanged. The change in the distribution of T cell subsets in the spleen and blood occurred in 2 months old castrated mice, as in 10 months old animals. P388 tumor grew better in post-pubertal and in castrated mice than in young mice. The intact mice survived longer than the castrated ones. The relative number of CD4+, CD8+ and CD2+ splenocytes was lower in transplanted intact mice than that in controls. The CD8+ and CD2+ subsets in the blood of 2 months transplanted mice were higher than those in controls, whereas in PBL, in 10 months old and castrated mice, the T lymphocyte subsets remain unchanged. Depo-testosterone (DT) injection strongly reduced weight and tumor growth in all the intact and castrated animals. A significant correlation is observed between the tumor weight and testosterone level in the plasma of the 2 months old DT treated mice. Moreover, DT injection induced a significant increase in the percentage of blood CD8+ cells in all the batches. These data indicate that physiologically, androgens affect the age-related distribution of lymphocyte T subsets and suggest that they slow down tumor growth, besides causing a direct effect, through an immunological process. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: T cell subsets; Leukemia cells; Age; Castration; Testosterone; Flow cytometry
1. Introduction There has been growing interest over the past few years in the changes that occur in the immune system during aging. The immune potentiality of animals declines with advancing age, with a decrease in most aspects of T cell responsiveness (Kay and Makinodan, 1981). The animal and human lymphocyte subsets differ markedly according to an individual’s age and sex (Goto and Nishioka, 1989). Numerous studies have demonstrated a deficiency in the ability of spleen cells from aged mice to comply a generation of cytotoxic cells to antigenic stimulation (Gotessman et al., 1988). The works of Lin et al. (1998) have shown that, the percentage of naive T cells declines with time, but the percentage of memory T * Corresponding author. Tel.: +33-4-66-23-28-06; fax: +33-4-66-23-49-57. E-mail address: [email protected] (J. Bureau). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 2 4 8 - 4 9 0 0 ( 0 2 ) 0 1 2 2 1 - 2
cells increases with age. Antigens presenting cells (APCs) from old mice induced a similar lymphocyte proliferative response, but lower lymphocyte cytotoxicity in comparison with APCs from young animals (Domini et al., 2002). The thymus also gradually regresses with age (Miller, 1979). The involution of the human thymus at puberty has been attributed to androgenic steroids (Bellamy et al., 1976). Several reports suggest that the immune system is influenced by sex hormones (Eidinger and Garett, 1972). The sex hormones influence thymocyte differentiation indirectly via their receptor within thymic epithelial cells (Kawashima et al., 1990). These hormones diffuse across the plasma membrane of target cells and bind to intracellular receptor proteins. The sex hormone–receptor complexes then bind to nuclear chromatins and regulate gene expression (Carson-Jurica et al., 1990), with the subsequent synthesis of proteins such as thymulin and thymosins (Seiki et al. 1988; Kawashima et al., 1991).
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Table 1 Age-related changes in plasma testosterone concentration (nmol/l), thymus, spleen (mg/100 g) and tumor (mg) weights of intact and castrated mice with or without DT treatment. The values are the means ± SEM. Each group contained 10 mice. The intact old (10 months) mice and castrated mice were compared to intact 2 month-old mice. The transplanted mice were compared to intact mice of the same age. The castrated transplanted mice were compared to castrated mice without tumors. The transplanted DT treated mice were compared to grafted untreated mice. TM: intact or castrated transplanted mice, DT: intact or castrated transplanted mice treated with DT
2 month-old
Intact TM DT
Castrated 2 month-old
Castrated TM DT
10 month-old
Intact TM DT
Thymus mg/100 g ± SEM 246 ± 68.1 200.1 ± 46.1 86.9 ± 36.8 P < 0.001 330.1 ± 36.8 P < 0.001 300.0 ± 25.5 170.0 ± 50.5 P < 0.001 110.3 ± 31.9 P < 0.001 102.2 ± 23.6 70.8 ± 24.6 P < 0.001
Spleen mg/100 g ± SEM 336.5 ± 24.3 437.1 ± 68.9 P < 0.01 508.9 ± 17.6 384.2 ± 47.9 400.5 ± 30.8 470.9 ± 60.0 335.5 ± 83.1 391.0 ± 34.3 450.5 ± 55.2
The immune system plays an important role in controlling tumor cell growth (Boon et al., 1994). Immunosenescence is believed to be one possible reason for increased incidence of most types of cancer in aging humans and animals (Anisinov, 1987). There is now good clinical and experimental evidence indicating a difference in the growth rate of primary tumors and in the incidence of metastases with age (Ershler et al., 1984). Some tumors are more specific to females than males and vice versa. The sex hormones are known to play an important role in the process of carcinogenesis (Furukawa et al., 1983) and in the growth and metastasis of cancer (Proctor et al., 1976). Several results also suggest the possible importance of sex steroids in the control of the proliferation of leukemic cells (Danel et al., 1985). However, the mechanism implicated in this regulation was not clear. We have therefore investigated the influence of age (pubertal and post-pubertal old) and androgen hormones on the distribution of T cell subsets in healthy mice and those harboring transplanted leukemia cells. 2. Results 2.1. Effects of age and castration 2.1.1. Organs weights (Table 1) The weight of the seminal vesicles was determined in intact 2 month-old mice (371.5 ± 63.2 mg/100 g) and in castrated mice (228 ± 67.9 mg/100 g, P < 0.01) to prove castration efficiency. The weight of the thymus decreased significantly in 10 month-old mice (P < 0.001) compared to 2 month-old mice, whereas it increased in castrated mice (P < 0.001).
Testosterone nmol/l ± SEM 22.4 ± 1 20.5 ± 2
Tumor weight mg ± SEM
Tumor appearance (%)
Survival (%)
9.5 ± 2.8
63.3
85
50.8 ± 11.1 P < 0.01 0.03 ± 0.01 P < 0.001 0.02 ± 0.008 20.5 ± 7.5 P < 0.001 0.6 ± 0.09 P < 0.001 0.5 ± 0.04 28.2 ± 5.2 P < 0.001
6.5 ± 2.3 P < 0.05
8.3
83.3
12.5 ± 4.5 8.5 ± 3.5
70 40
50 40
11.2 ± 4.3 7.9 ± 3.3
71.4 30
85.7 60
No significant change was observed in the weight of the spleen with age or castration (Table 1). 2.1.2. Serum testosterone (Table 1) The plasma testosterone level decreased significantly with age and castration (P < 0.001). 2.1.3. Analysis of T cell subsets in the spleen and blood 2.1.3.1. Blood (Fig. 1). There were significantly more circulating CD2+ (P < 0.05) and CD4+ cells (P < 0.005) in 2 month-old mice than in 10 month-old mice. A moderately, but not significantly, lower percentage of CD4+ and CD2+ cells was observed in castrated mice. The ratio CD4+/CD8+ was significantly lower in castrated (P < 0.05) and aged mice (10 months P < 0.01) than in intact 2 month-old mice). 2.1.3.2. Spleen (Figs. 2 and 3a). Castration reduced the number of CD4+, CD2+ (P < 0.01) and CD8+ (P < 0.05) cells in the spleen. The percentage of CD4+, CD8+ (P < 0.05) and CD2+ (P < 0.01) was also significantly lower in old mice compared to 2 month-old animals. There was a correlation between the percentage of CD8+ (r = 0.95, P < 0.01), CD4+ (r = 0.7, P < 0.05) (Fig. 3a) and plasma testosterone concentration level in 10 month-old mice. 2.2. Tumor growth 2.2.1. Tumor weight (Table 1) Though there was a decrease in the tumor weight in young mice compared to the 10 month-old and castrated animals, no significant difference was observed.
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Fig. 1. Age-related changes in the percentages of CD4+ (a), CD8+ (b), CD2+ (c) and CD4+ /CD8+ ratio (d) in the PBL of intact and castrated mice with or without tumor cells, whether treated or not with DT. Data are means ± SEM. C P < 0.05; CC P < 0.01 compared to intact 2 month-old mice. + P < 0.05; ++ P < 0.01 grafted mice compared to non-transplanted animals. * P < 0.05; ** P < 0.01 grafted mice treated with DT compared to mice bearing tumor without DT treatment.
2.2.2. Tumor growth (Table 1)
2.2.3. Organs weight (Table 1)
The percentage of mice that developed tumors was high in old mice (71.4%) and in castrated mice (70%). Only 63.3% of the young mice developed tumors. The intact mice (2 and 10 month-old) survived longer (85%) than castrated mice (50%).
The thymus weight of the transplanted mice and control mice of the same age were not significantly different. The weight of the spleen was significantly higher in 2 month-old transplanted mice than in controls (P < 0.01).
Fig. 2. Age-related changes in the percentages of CD4+ (a), CD8+ (b), and CD2+ (c) in the spleen of intact and castrated mice with or without tumor cells, whether treated or not with DT. Data are means ± SEM. C P < 0.05; CC P < 0.01 compared to intact 2 month-old mice. + P < 0.05; ++ P < 0.01. Grafted mice compared to non-transplanted animals. * P < 0.05; ** P < 0.01 grafted mice treated with DT compared to mice bearing tumor without DT treatment.
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Fig. 3. Correlation between plasma testosterone level and a) tumor weight in DT treated 2 month-old mice; b) the percentage of CD8+, CD4+ cells in the spleen of 10 month-old mice.
Fig. 4. Correlation between tumor weight and a) the percentage of CD8+, CD4+, and CD2+ cells in the blood; b) the percentage of CD4+ splenocytes.
2.2.4. T cell subsets
2.3. Effect of depo-testosterone treatment
2.2.4.1. Blood (Figs. 1 and 4a). The percentage of CD8+ (P < 0.05) and CD2+ (P < 0.01) cells in grafted 2 month-old mice was significantly higher than in control mice of the same age. There was no significant difference in 10 monthold mice bearing tumor and the controls. The percentage of CD4+ cells (P < 0.05) in castrated mice with tumor was greater than in that of control castrated mice. The CD4+/CD8+ ratio was lower (P < 0.01) in 2 month-old mice with tumor than that in the control. A positive correlation was observed in 2 month-old mice between the tumor weight and the percentage of CD8+ (r = 0.74, P < 0.05), CD4+ (r = 0.88, P < 0.01) and CD2+ cells (r = 0.82, P < 0.05) (Fig. 4a).
2.3.1. Tumor weight (Table 1)
2.2.4.2. Spleen (Figs. 2 and 4b) There was a positive correlation in tumor weight and percentage of CD4+ cells (r = 0.8, P < 0.05, Fig. 5) in 2 month-old mice (Fig. 4b). The percentage of CD4+, CD8+ (P < 0.01) and CD2+ (P < 0.05) cells was lower in 2 monthold mice transplanted with tumors than that in healthy controls. There was a decrease only in the percentage of CD4+ cells (P < 0.05) in 10 month-old mice with tumors compared to 10 month-old mice without tumors. There was no significant difference between castrated mice with tumors and castrated mice without tumors.
Tumor weight is lower in DT treated than that in nontreated mice. 2.3.2. Tumor growth (Table 1) DT injection strongly decreased tumor growth in all mice (2, 10 months and castrated animals) compared to the noninjected animals. DT injection did not modify the mortality level in 2 month-old mice, but slightly increased the mortality in 10 month-old control and castrated animals. 2.3.3. Plasma testosterone (Table 1, Fig. 3b) Plasma testosterone level increased after DT injection in the three groups of young (P < 0.01) mice, old and castrated (P < 0.001) animals. The regressive curve is significant between tumor weight and testosterone level (r = 0.9, P < 0.05, Fig. 3b). 2.3.4. Organs weight 2.3.4.1. Seminal vesicles. DT treatment induced a significant (P < 0.001) increase in the weight of seminal vesicles in intact (371.5 ± 63.2 to 514.6 ± 72 mg) and castrated mice (228 ± 67.9 to 480.3 ± 76.3 mg) compared to the controls.
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2.3.4.2. Thymus and spleen (Table 1). An important decrease in thymus weight was observed in all the batches under DT treatment. There was no change in spleen weight. 2.3.5. Lymphocyte subpopulations 2.3.5.1. Blood (Fig. 1). DT injection increased the percentage of CD8+ cells in 2 month-old mice (P < 0.05), 10 monthold and castrated animals (P < 0.01). The percentage of CD4+ cells increased (P < 0.05) in two sets of animals, the 10 month-old and the castrated ones with DT, but not in 2 month-old mice. 2.3.5.2. Spleen (Fig. 2). There was an increase in the number of CD8+ cell population in DT treated tumor bearing mice than that in untreated animals (P < 0.05). 3. Discussion The purpose of this work was to study the influence of androgens’ physiological modifications (pubertal and postpubertal age), as well as the effect of castration and testosterone on T-lymphocyte subsets in healthy and leukemia grafted mice. The data from the first part of the study show that the distribution of splenocyte and peripheral blood lymphocyte (PBL) subsets changes with age. After 3 weeks of castration, the androgen deprivation had the same effect on the distribution of the lymphocytes in spleen and PBL as in post-pubertal old mice. The percentage of CD4+ splenocyte and PBL decreased significantly with age, as did the CD4+/CD8+ ratio. But the percentage of CD8+ cells in the PBL remained unchanged with advancing age. Previous studies have shown that the number of CD8+ cells in the peripheral blood mouse strains (CBA and C57BL) remains relatively unchanged during the entire lifespan (Boersma et al., 1985), but others have reported that the percentage of CD2+ and CD8+ cells in the spleen increases with age (Utsuyama and Hirokawa, 1987). The diversity of age-related changes in various CD4+ and CD8+ cell subsets reflects the great polymorphism of the process. Mice of different strains can be divided into groups distinguished by differences in the age-related changes in various functional types of T cells (Dubiski et al., 1989). The recent work of Boon et al. (2002) has reported that the lytic capacity of CD8+ cells, after in vitro stimulation of peripheral blood mononuclear cells with influenza A virus, seems lower in 68–70 year-old donors than that in 18–20 year-old donors. The percentage of CD4+ cells remains unchanged from infancy to adolescence, but the percentage of CD8+ T cells was lowest at birth and reached a maximal level within a 1–2 year period (Lin et al., 1998). However, no comparative studies have been conducted to determine whether the physiological influences due to sex hormone differences affect age-related changes in the immune system.
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We also investigated the impact of transient changes in the sex hormone profile at puberty (2 months), and post-puberty (10 months) in intact mice as well as the androgens’ deprivation of castrated mice. Preliminary work (Aboudkhil, 1991) has shown that plasma testosterone concentration in BDF1 4 month-old mice decreases and that it reaches a minimum level at 10 months. The C57BL/6 mice are considered to be sexually and immunologically mature (Rao et al., 1996) at 2–3 months. Compared to the control animals, post-pubertal old mice and castrated mice had fewer CD4+, CD8+ and CD2+ cells in the spleen. Testosterone injections are known (Aboudkhil et al., 1991) to be involved in the distribution of some T lymphocyte subsets, at least in CD4+ and CD8+ thymocytes and splenocytes. As the relative numbers of mature T cells (CD4+ CD3+ and CD8+ CD3+) were decreased in the spleens of castrated animals (Viselli et al., 1995), a reverse effect was observed in testosterone treated mice. The number of CD2+ and CD8+ cells in the spleen increases gradually after birth to reach a plateau at around 3 months of age, and decreases after 12 months of age (Utsuyama and Hirokawa, 1987). Testosterone concentration is high at birth and then decreases (Murad and Haynes, 1985). In our results, there was a correlation between the proportion of CD4+ (r = 0.7, P < 0.05), and CD8+ (r = 0.95, P < 0.01) in the spleen and the plasma testosterone concentration in BDF1 mice. The decrease in spleen T lymphocyte subsets (CD4+, CD8+ and CD2+) and in the blood (CD4+ and CD2+) in 10 month-old and castrated animals could be related to the decrease in the plasma testosterone. Our data suggest that androgens have a physiological effect on the age-related distribution of lymphocyte T subsets. We also examined the influence of physiological (pubertal and post-pubertal age) androgens, castration and DT treatment on the murine leukemia P388 development. The weight and the rate of tumor growth were higher in post-pubertal old and castrated mice than those in young mice. The relative number of CD4+, CD8+ and CD2+ splenocytes in grafted DT untreated mice was low. However, CD8+ and CD4+ splenocytes in the blood of 2 month-old tumor bearing mice were more numerous. The incidence of most types of cancer is higher in aging humans and animals. The high rate of tumor development in old mice can be explained by the immune deficient state associated with aging (Anisinov, 1987). The percentage of CD2+ and CD8+ in the spleen cells of mice with large tumors appeared to be much lower than the percentages in normal mice and mice with a small tumor. The responses of the spleen cells of mice with tumors to three mitogens (PHA, ConA, and LPS) had considerably reduced (Buessow et al., 1984). In fact, in young mice, the decrease of T lymphocyte subsets observed in the spleen was counterbalanced by a
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subset increase (CD4+ and CD8+) in PBL, whereas in 10 month-old and castrated mice, the T lymphocyte subsets of PBL remained unchanged. The lymphocyte cytotoxicity activity of CD8+ cells seems to be important and a crucial effector system for inhibiting tumor growth in vivo (Geissler et al., 1997). As the number of CD8+ cells in the blood of 2 month-old mice is high, it could explain the difficulty for the tumor to develop. DT injection highly decreases the percentage of tumor bearing mice in young, old and castrated ones. Over 30 d, 40% of the old and 60% of the castrated mice died; on the other hand, 16.7% of mortality was found in younger ones. Moreover, testosterone inhibits the in vivo tumorigenic properties of the 1246-3A cells. Castrated male mice receiving injections of 1246-3A cells developed a larger tumor at a higher frequency than sham-operated animals. The administration of testosterone to castrated male mice resulted in a dramatic decrease in tumor development (Serrero et al., 1992). Cross and Dexter (1991) show that testosterone may inhibit tumor growth in vivo by modifying vascularization, which is necessary for tumor development. The decrease in serum level of dehydroepiandrosterone may be associated with patients who have some clinical subtypes of adult T-cell leukemia (ATL) (Kimiharu et al., 1996). Testosterone can inhibit the proliferation and the clonogenic potential of the human monoblastic leukemic cell line U937. The growth inhibition was associated with cell cycle arrest, U937 cells accumulating in G2/M phase (Mossuz et al., 1998). In addition, the inhibition of tumor growth was strongly associated with the level of CTL activity present in CD8 cells derived from the spleen. Moreover, the level of CD8+ CTL activity directly correlated with the degree of inhibition of tumor growth (Serrero et al., 1992). Our results show that CD8+ cells increased in the three batches of animals under DT treatment. A positive correlation was found between the percentage of blood CD8+ cells, CD4+ of spleen cells and tumor weight. Thus, we suggest, besides the direct effect on malignant cells, testosterone can inhibit tumoral growth by modifying CD8+ and CD4+ cells distribution. Recent work (Segal et al., 2002) demonstrated that IL-10 producing CD4+ T cells can manifest anti-tumor functions. The IL-10 maintains the number of anti-tumor CD8+ T cells. Liva and Voskuhl (2001) showed that testosterone can act directly via androgen receptor on CD4+ T lymphocyte to increase IL-10 gene expression. The testosterone action on the growth inhibition may act through CD4+ and CD8+ cells and their cytokine mediators. Our results support other researchers’ propositions (Mossuz, 1998; Blagosklonny and Neckers, 1994; Kimiharu et al., 1996) about the fact that sexual hormones could be used as an adjuvant to improve the chemotherapeutic activity of an anti-tumor agent in leukemia patients.
4. Materials and methods 4.1. Animals BDF1 male mice (C57BL/6 × DBA/2)F1, 3 weeks old, and 10 month-old (IFFA-CREDO, Les Oncins, France), were housed in an air conditioned room (temperature 21 ± 2 °C) in standard cages housing five animals each. Food and water were available ad libitum. The mice were kept on a 12 h light–dark cycle (LD 12:12 h). 4.2. Castration Male mice were castrated at the age of 4 weeks. They were anesthetized with ether and castrated by making an abdominal incision, removing the testes and closing the incision with 6-0 silk sutures. As controls, there were two groups: shamoperated and non-operated (intact) animals. It was found that there were no significant results between the sham-operated group and the non-operated controls; hence the data from these two groups were pooled. The castrated mice were 2 months old, when they were killed. Castration was tested only in 2 month-old mice. The 10 month-old mice were not castrated as their plasma testosterone level was low. 4.3. Steroids Depo-testosterone (DT, Testosterone 17beta-cypionate, SIGMA, St-Louis, MO, USA) in peanut oil (PNO) was administered subcutaneously in mice at a dose of 0.5 mg/100 g body weight, at a final volume of 0.2 ml. The same volume of PNO was injected into the castrated and intact control group. DT was administered to animals on alternate days for a period of 2 weeks. Steroids were injected into treated animals 1 week after castration. The dose, route and frequency of DT administration were chosen based on the work of Ahmed et al. (1985). 4.4. Transplanted mice P388 tumor cell line (from mouse DBA/2 monocyte–macrophage lymphoma) was obtained from Flow, France. They were maintained in culture in Fisher medium supplemented with 5% fetal bovine serum, 1% glutamine (2 mM) and 1% penicillin–streptomycin, at 37 °C in a humidified atmosphere of 5% CO2. The cells were counted with a Coulter counter (ZB, Coulter, Margency, France). Trypan blue exclusion indicated that the viability exceeded 90%. The cells were injected subcutaneously at 105 cells per mouse and the mice were examined daily to monitor survival and tumor growth. Mice were divided into two batches and killed after 15 d or 30 d after tumor grafting. The P388 tumors could only be followed for approximately 2 weeks after which the animals developed leukemia and died (Aabo et al., 1989). Control mice were injected with Fisher medium, or with PNO. No difference was observed between the two batches.
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4.5. Serum testosterone Mice were killed by cardiac exsanguination. The blood was collected in heparinized syringes and the plasma was separated by centrifugation. The testosterone concentration in the plasma was measured by radio-immunoassay by Dr. G. Baudin (Department of Nuclear Medicine, CHU-Nîmes).
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4.9. Statistical analysis The data were analyzed with Student’s t-test and standard correlation. A P value of less than 0.05 was considered significant. The data were plotted using Cricket Graph software.
Acknowledgements 4.6. Cell preparation The thymus and spleen were removed from each mouse and weighed. The weight of the organs was expressed as mg/100 g body weight. The spleen was placed in individual Petri dishes containing phosphate-buffered saline (PBS). Single-cell suspensions were prepared and the cells were washed twice in cold PBS containing 2% bovine serum albumin, 0.01 % sodium azide, and later resuspended in an appropriate volume. Erythrocytes were lyzed by adding 2 ml of lyzing solution to spleen cells and blood samples. The cells were counted in a Coulter counter. The viability was determined by trypan blue exclusion.
4.7. T cell phenotype studies Each sample of lymphoid cells was labeled by incubation with 4 µl monoclonal antibody (MoAb) conjugated to fluorescein (FITC) plus 4 µl MoAb conjugated to phyco-erythrin (PE) for 20 min at 4 °C. The samples were then washed in cold PBS and the cells suspended in 1 ml PBS. The MoAbs used for the phenotypic analysis were thy 1.2-FITC (CD2, clone 30H12, Becton Dickinson, Mountain View, CA, USA), which recognizes immature and mature T cells; Lyt-2-FITC (CD8, Clone 53-6.7, Becton Dickinson) which recognizes cytotoxic/suppressor cells; and L3T4 (CD4, Clone GK1,5, Becton Dickinson) which recognizes helper/inducer cells.
4.8. Flow cytometry analysis The T cell phenotype study was performed using a flow cytometer FACScan (Becton-Dickinson, Mountain View, CA, USA) equipped with an Innova 90-5 (Coherent, Palo alto, CA, USA) argon ion laser operating at 488 nm and 515 mW in light-regulated mode. Light scattering data and fluorescence parameters were collected by user-defined protocols and stored in list mode via Lysis II program. The phenotypic analysis, considering the proportion of positive cells, was determined from histograms, and the red (for L3T4) and green (for Lyt-2 and Thy1,2) fluorescences were used to determine the number of cells per channel based on 20,000 events gated in the lymphocyte area of cytograms: forward angle scatter vs. right angle scatter.
We thank Dr. G. Baudin for the dosage of testosterone in the Department of Nuclear Medicine. This work was supported by a grant from Ligue nationale contre le cancer (comité du gard et de l’aude).
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Dubiski, S., Ponnapan, U., Cinader, B., 1989. Strain polymorphism in progression of aging: changes in CD4, CD8 bearing subpopulations. Immunol. Lett 23, 1–8. Eidinger, D., Garett, T., 1972. Studies of the regulatory effects of the sex hormones on antibody formation and stem cell differentiation. J. Exp. Med 136, 1089. Ershler, B.W., Stewart, J.A., Hacker, M.P., Moore, A.L., Tindle, B.H., 1984. B16 murine melanoma and aging: slower growth and longer survival in old mice. J. Natl. Cancer Inst 72, 161–164. Furukawa, H., Iwanaga, T., Ichikawa, H., 1983. Effect of sex hormones on carcinogenesis of experimental gastric cancer. A chronological study. Jpn. J. Digest. Tract 8, 1410–1414. Geissler, M., Wands, G., Gesien, A., Delamonte, S., Bellet, D., Wands, J.R., 1997. Genetic immunization with the free human chorionic gonadotropin beta-subunit elicits cytotoxic T lymphocyte responses and protects against tumor formation in mice. Lab. Invest 76, 859–871. Gotessman, S., Edington, J.M., Thobecke, G.J., 1988. Proliferative and cytotoxic immune functions in aging mice: IV effects of suppressor cell population from aged and young mice. J. Immunol 140, 1783–1790. Goto, M., Nishioka, K., 1989. Age and sex related changes of the lymphocyte subsets in healthy individuals: an analysis by two dimensional flow cytometry. J. Gerontol 44 (51). Kawashima, I., Sakabe, K., Seiki, K., Fujii-Hanamoto, H., 1990. Hormone and immune response, with special reference to steroid hormone 3. Sex steroid effect on T-cell differentiation. Tokai J. Exp.Clin. Med 15, 213–218. Kawashima, I., Sakabe, K., Seiki, K., Fujii-Hanamoto, H., Akatsuka, A., Tsukamoto, H., 1991. Localisation of sex steroid receptor cells, with special reference to thymulin (FTS)-producing cells in female rat thymus. Thymus 18, 79–93. Kay, M., Makinodan, T., 1981. Relationship between aging and the immune system. Prog. Allergy 29, 134–143. Kimiharu, U., Toshiaki, U., Maki, O., Satoko, N., Yoshifusa, T., Mashito, I., Shuichi, H., Teruktsu, A., 1996. Serum dehydroepiandrosterone and DHEA-sulfate in patients with adult T-cell leukemia and human T-lymphotropic virus type I carriers. Am. J. Hematol 53, 165–168.
Lin, S.C., Chou, C.C., Tsai, M.J., Wu, K.H., Huang, M.T., Wang, L.H., Chiang, B.L., 1998. Age-related changes in blood lymphocyte subsets of Chinese children. Pediatr. Allergy Immunol 9, 215–220. Liva, S.M., Voskuhl, R.R., 2001. Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production. J. Immunol. 167 (4), 2060–2067. Miller, J.F., 1979. Experimental thymology has come of age. Thymus 1 (3). Mossuz, P., Cousin, F., Castinel, A., Chauvet, M., Sotto, M.F., Polack, B., Sotto, J.J., Kolodie, L., 1998. Effects of two sex steroids (17 alphaestradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937. Leuk. Res 22, 1063–1072. Murad, F., Haynes, F., 1985. In: Goodman, Alfred, Gilman, Louis, Goodman, Rall, Theodore W., Murad, Ferid (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutic, seventh ed. pp. 1440–1456. Proctor, J.W., Auclair, B.G., Stokowski, L., 1976. Endocrine factors and the growth and spread of B16 melanoma. J. Natl. Cancer Inst 57, 1197–1198. Rao, L.V., Cleveland, R.P., Kimmel, R.J., Ataya, K.M., 1996. Gonadotropinreleasing hormone agonist influences absolute levels of lymphocyte subsets in vivo in male mice. Immunol. Cell Biol 74, 137–143. Seiki, K., Sakabe, K., Fujii-Hanamoto, H., Kawashima, I., Ogawa, A., Akatsuka, A., Yanaihara, N., 1988. Localization of sex steroid receptor cells in thymus, with special reference to thymic factor-producing cells. Med. Sci. Res 16, 967–970. Segal, B.M., Glass, D.D., Shevach, E.M., 2002. Cutting edge: IL-10producing CD4+ T cells mediate tumor rejection. J. Immunol. 168 (1), 1–4. Serrero, G., Lepak, N.M., Hayashi, J., Eisenger, D.P., 1992. Effect of testosterone on the growth properties and on epidermal growth factor receptor expression in the teratoma-derived tumorigenic cell line 12463A. Cancer Res 52, 4242–4247. Utsuyama, M., Hirokawa, K., 1987. Age-related changes of splenic T cells in mice – a flow cytometric analysis. Mechs. Ageing Dev 40, 89–102. Viselli, S.M., Stanziale, S., Shilts, K., Kovacs, W.J., Olsen, N., 1995. Castration alters peripheral immune function in normal male mice. Immunology 84, 337–342.
Influence of age, castration, and testosterone on T cell subsets in healthy and leukemia grafted mice Souad Aboudkhil a,b,c, Abdelhamid Zaîd b, Laurent Henry c, Jean-Paul Bureau c,* a
Ufr environnement et santé, Department of Biology, Faculty of Science and Technique, University Hassan II, Quartier Yasmina, BP 146, Mohammedia, Morocco b Ufr environnement et santé, Department of Biology, Faculty of Science, University Moulay Ismaîl, BP 4010, Beni M’hamed, 50 006 Meknes, Morocco c Laboratory of Cell Biology and Molecular Cytogenetics, Faculty of Medicine, Montpellier-Nîmes, Avenue Kennedy, 30900 Nîmes, France Received 2 May 2002; accepted 12 December 2002
Abstract The distribution of T cell subsets in pubertal (2 months) and post-pubertal (10 months) mice showed a significant decrease in the percentage of CD4+ splenocytes and peripheral blood lymphocytes (PBL) with age, unlike the percentage of CD8+ cells in PBL, which remained unchanged. The change in the distribution of T cell subsets in the spleen and blood occurred in 2 months old castrated mice, as in 10 months old animals. P388 tumor grew better in post-pubertal and in castrated mice than in young mice. The intact mice survived longer than the castrated ones. The relative number of CD4+, CD8+ and CD2+ splenocytes was lower in transplanted intact mice than that in controls. The CD8+ and CD2+ subsets in the blood of 2 months transplanted mice were higher than those in controls, whereas in PBL, in 10 months old and castrated mice, the T lymphocyte subsets remain unchanged. Depo-testosterone (DT) injection strongly reduced weight and tumor growth in all the intact and castrated animals. A significant correlation is observed between the tumor weight and testosterone level in the plasma of the 2 months old DT treated mice. Moreover, DT injection induced a significant increase in the percentage of blood CD8+ cells in all the batches. These data indicate that physiologically, androgens affect the age-related distribution of lymphocyte T subsets and suggest that they slow down tumor growth, besides causing a direct effect, through an immunological process. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: T cell subsets; Leukemia cells; Age; Castration; Testosterone; Flow cytometry
1. Introduction There has been growing interest over the past few years in the changes that occur in the immune system during aging. The immune potentiality of animals declines with advancing age, with a decrease in most aspects of T cell responsiveness (Kay and Makinodan, 1981). The animal and human lymphocyte subsets differ markedly according to an individual’s age and sex (Goto and Nishioka, 1989). Numerous studies have demonstrated a deficiency in the ability of spleen cells from aged mice to comply a generation of cytotoxic cells to antigenic stimulation (Gotessman et al., 1988). The works of Lin et al. (1998) have shown that, the percentage of naive T cells declines with time, but the percentage of memory T * Corresponding author. Tel.: +33-4-66-23-28-06; fax: +33-4-66-23-49-57. E-mail address: [email protected] (J. Bureau). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 2 4 8 - 4 9 0 0 ( 0 2 ) 0 1 2 2 1 - 2
cells increases with age. Antigens presenting cells (APCs) from old mice induced a similar lymphocyte proliferative response, but lower lymphocyte cytotoxicity in comparison with APCs from young animals (Domini et al., 2002). The thymus also gradually regresses with age (Miller, 1979). The involution of the human thymus at puberty has been attributed to androgenic steroids (Bellamy et al., 1976). Several reports suggest that the immune system is influenced by sex hormones (Eidinger and Garett, 1972). The sex hormones influence thymocyte differentiation indirectly via their receptor within thymic epithelial cells (Kawashima et al., 1990). These hormones diffuse across the plasma membrane of target cells and bind to intracellular receptor proteins. The sex hormone–receptor complexes then bind to nuclear chromatins and regulate gene expression (Carson-Jurica et al., 1990), with the subsequent synthesis of proteins such as thymulin and thymosins (Seiki et al. 1988; Kawashima et al., 1991).
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Table 1 Age-related changes in plasma testosterone concentration (nmol/l), thymus, spleen (mg/100 g) and tumor (mg) weights of intact and castrated mice with or without DT treatment. The values are the means ± SEM. Each group contained 10 mice. The intact old (10 months) mice and castrated mice were compared to intact 2 month-old mice. The transplanted mice were compared to intact mice of the same age. The castrated transplanted mice were compared to castrated mice without tumors. The transplanted DT treated mice were compared to grafted untreated mice. TM: intact or castrated transplanted mice, DT: intact or castrated transplanted mice treated with DT
2 month-old
Intact TM DT
Castrated 2 month-old
Castrated TM DT
10 month-old
Intact TM DT
Thymus mg/100 g ± SEM 246 ± 68.1 200.1 ± 46.1 86.9 ± 36.8 P < 0.001 330.1 ± 36.8 P < 0.001 300.0 ± 25.5 170.0 ± 50.5 P < 0.001 110.3 ± 31.9 P < 0.001 102.2 ± 23.6 70.8 ± 24.6 P < 0.001
Spleen mg/100 g ± SEM 336.5 ± 24.3 437.1 ± 68.9 P < 0.01 508.9 ± 17.6 384.2 ± 47.9 400.5 ± 30.8 470.9 ± 60.0 335.5 ± 83.1 391.0 ± 34.3 450.5 ± 55.2
The immune system plays an important role in controlling tumor cell growth (Boon et al., 1994). Immunosenescence is believed to be one possible reason for increased incidence of most types of cancer in aging humans and animals (Anisinov, 1987). There is now good clinical and experimental evidence indicating a difference in the growth rate of primary tumors and in the incidence of metastases with age (Ershler et al., 1984). Some tumors are more specific to females than males and vice versa. The sex hormones are known to play an important role in the process of carcinogenesis (Furukawa et al., 1983) and in the growth and metastasis of cancer (Proctor et al., 1976). Several results also suggest the possible importance of sex steroids in the control of the proliferation of leukemic cells (Danel et al., 1985). However, the mechanism implicated in this regulation was not clear. We have therefore investigated the influence of age (pubertal and post-pubertal old) and androgen hormones on the distribution of T cell subsets in healthy mice and those harboring transplanted leukemia cells. 2. Results 2.1. Effects of age and castration 2.1.1. Organs weights (Table 1) The weight of the seminal vesicles was determined in intact 2 month-old mice (371.5 ± 63.2 mg/100 g) and in castrated mice (228 ± 67.9 mg/100 g, P < 0.01) to prove castration efficiency. The weight of the thymus decreased significantly in 10 month-old mice (P < 0.001) compared to 2 month-old mice, whereas it increased in castrated mice (P < 0.001).
Testosterone nmol/l ± SEM 22.4 ± 1 20.5 ± 2
Tumor weight mg ± SEM
Tumor appearance (%)
Survival (%)
9.5 ± 2.8
63.3
85
50.8 ± 11.1 P < 0.01 0.03 ± 0.01 P < 0.001 0.02 ± 0.008 20.5 ± 7.5 P < 0.001 0.6 ± 0.09 P < 0.001 0.5 ± 0.04 28.2 ± 5.2 P < 0.001
6.5 ± 2.3 P < 0.05
8.3
83.3
12.5 ± 4.5 8.5 ± 3.5
70 40
50 40
11.2 ± 4.3 7.9 ± 3.3
71.4 30
85.7 60
No significant change was observed in the weight of the spleen with age or castration (Table 1). 2.1.2. Serum testosterone (Table 1) The plasma testosterone level decreased significantly with age and castration (P < 0.001). 2.1.3. Analysis of T cell subsets in the spleen and blood 2.1.3.1. Blood (Fig. 1). There were significantly more circulating CD2+ (P < 0.05) and CD4+ cells (P < 0.005) in 2 month-old mice than in 10 month-old mice. A moderately, but not significantly, lower percentage of CD4+ and CD2+ cells was observed in castrated mice. The ratio CD4+/CD8+ was significantly lower in castrated (P < 0.05) and aged mice (10 months P < 0.01) than in intact 2 month-old mice). 2.1.3.2. Spleen (Figs. 2 and 3a). Castration reduced the number of CD4+, CD2+ (P < 0.01) and CD8+ (P < 0.05) cells in the spleen. The percentage of CD4+, CD8+ (P < 0.05) and CD2+ (P < 0.01) was also significantly lower in old mice compared to 2 month-old animals. There was a correlation between the percentage of CD8+ (r = 0.95, P < 0.01), CD4+ (r = 0.7, P < 0.05) (Fig. 3a) and plasma testosterone concentration level in 10 month-old mice. 2.2. Tumor growth 2.2.1. Tumor weight (Table 1) Though there was a decrease in the tumor weight in young mice compared to the 10 month-old and castrated animals, no significant difference was observed.
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Fig. 1. Age-related changes in the percentages of CD4+ (a), CD8+ (b), CD2+ (c) and CD4+ /CD8+ ratio (d) in the PBL of intact and castrated mice with or without tumor cells, whether treated or not with DT. Data are means ± SEM. C P < 0.05; CC P < 0.01 compared to intact 2 month-old mice. + P < 0.05; ++ P < 0.01 grafted mice compared to non-transplanted animals. * P < 0.05; ** P < 0.01 grafted mice treated with DT compared to mice bearing tumor without DT treatment.
2.2.2. Tumor growth (Table 1)
2.2.3. Organs weight (Table 1)
The percentage of mice that developed tumors was high in old mice (71.4%) and in castrated mice (70%). Only 63.3% of the young mice developed tumors. The intact mice (2 and 10 month-old) survived longer (85%) than castrated mice (50%).
The thymus weight of the transplanted mice and control mice of the same age were not significantly different. The weight of the spleen was significantly higher in 2 month-old transplanted mice than in controls (P < 0.01).
Fig. 2. Age-related changes in the percentages of CD4+ (a), CD8+ (b), and CD2+ (c) in the spleen of intact and castrated mice with or without tumor cells, whether treated or not with DT. Data are means ± SEM. C P < 0.05; CC P < 0.01 compared to intact 2 month-old mice. + P < 0.05; ++ P < 0.01. Grafted mice compared to non-transplanted animals. * P < 0.05; ** P < 0.01 grafted mice treated with DT compared to mice bearing tumor without DT treatment.
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Fig. 3. Correlation between plasma testosterone level and a) tumor weight in DT treated 2 month-old mice; b) the percentage of CD8+, CD4+ cells in the spleen of 10 month-old mice.
Fig. 4. Correlation between tumor weight and a) the percentage of CD8+, CD4+, and CD2+ cells in the blood; b) the percentage of CD4+ splenocytes.
2.2.4. T cell subsets
2.3. Effect of depo-testosterone treatment
2.2.4.1. Blood (Figs. 1 and 4a). The percentage of CD8+ (P < 0.05) and CD2+ (P < 0.01) cells in grafted 2 month-old mice was significantly higher than in control mice of the same age. There was no significant difference in 10 monthold mice bearing tumor and the controls. The percentage of CD4+ cells (P < 0.05) in castrated mice with tumor was greater than in that of control castrated mice. The CD4+/CD8+ ratio was lower (P < 0.01) in 2 month-old mice with tumor than that in the control. A positive correlation was observed in 2 month-old mice between the tumor weight and the percentage of CD8+ (r = 0.74, P < 0.05), CD4+ (r = 0.88, P < 0.01) and CD2+ cells (r = 0.82, P < 0.05) (Fig. 4a).
2.3.1. Tumor weight (Table 1)
2.2.4.2. Spleen (Figs. 2 and 4b) There was a positive correlation in tumor weight and percentage of CD4+ cells (r = 0.8, P < 0.05, Fig. 5) in 2 month-old mice (Fig. 4b). The percentage of CD4+, CD8+ (P < 0.01) and CD2+ (P < 0.05) cells was lower in 2 monthold mice transplanted with tumors than that in healthy controls. There was a decrease only in the percentage of CD4+ cells (P < 0.05) in 10 month-old mice with tumors compared to 10 month-old mice without tumors. There was no significant difference between castrated mice with tumors and castrated mice without tumors.
Tumor weight is lower in DT treated than that in nontreated mice. 2.3.2. Tumor growth (Table 1) DT injection strongly decreased tumor growth in all mice (2, 10 months and castrated animals) compared to the noninjected animals. DT injection did not modify the mortality level in 2 month-old mice, but slightly increased the mortality in 10 month-old control and castrated animals. 2.3.3. Plasma testosterone (Table 1, Fig. 3b) Plasma testosterone level increased after DT injection in the three groups of young (P < 0.01) mice, old and castrated (P < 0.001) animals. The regressive curve is significant between tumor weight and testosterone level (r = 0.9, P < 0.05, Fig. 3b). 2.3.4. Organs weight 2.3.4.1. Seminal vesicles. DT treatment induced a significant (P < 0.001) increase in the weight of seminal vesicles in intact (371.5 ± 63.2 to 514.6 ± 72 mg) and castrated mice (228 ± 67.9 to 480.3 ± 76.3 mg) compared to the controls.
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2.3.4.2. Thymus and spleen (Table 1). An important decrease in thymus weight was observed in all the batches under DT treatment. There was no change in spleen weight. 2.3.5. Lymphocyte subpopulations 2.3.5.1. Blood (Fig. 1). DT injection increased the percentage of CD8+ cells in 2 month-old mice (P < 0.05), 10 monthold and castrated animals (P < 0.01). The percentage of CD4+ cells increased (P < 0.05) in two sets of animals, the 10 month-old and the castrated ones with DT, but not in 2 month-old mice. 2.3.5.2. Spleen (Fig. 2). There was an increase in the number of CD8+ cell population in DT treated tumor bearing mice than that in untreated animals (P < 0.05). 3. Discussion The purpose of this work was to study the influence of androgens’ physiological modifications (pubertal and postpubertal age), as well as the effect of castration and testosterone on T-lymphocyte subsets in healthy and leukemia grafted mice. The data from the first part of the study show that the distribution of splenocyte and peripheral blood lymphocyte (PBL) subsets changes with age. After 3 weeks of castration, the androgen deprivation had the same effect on the distribution of the lymphocytes in spleen and PBL as in post-pubertal old mice. The percentage of CD4+ splenocyte and PBL decreased significantly with age, as did the CD4+/CD8+ ratio. But the percentage of CD8+ cells in the PBL remained unchanged with advancing age. Previous studies have shown that the number of CD8+ cells in the peripheral blood mouse strains (CBA and C57BL) remains relatively unchanged during the entire lifespan (Boersma et al., 1985), but others have reported that the percentage of CD2+ and CD8+ cells in the spleen increases with age (Utsuyama and Hirokawa, 1987). The diversity of age-related changes in various CD4+ and CD8+ cell subsets reflects the great polymorphism of the process. Mice of different strains can be divided into groups distinguished by differences in the age-related changes in various functional types of T cells (Dubiski et al., 1989). The recent work of Boon et al. (2002) has reported that the lytic capacity of CD8+ cells, after in vitro stimulation of peripheral blood mononuclear cells with influenza A virus, seems lower in 68–70 year-old donors than that in 18–20 year-old donors. The percentage of CD4+ cells remains unchanged from infancy to adolescence, but the percentage of CD8+ T cells was lowest at birth and reached a maximal level within a 1–2 year period (Lin et al., 1998). However, no comparative studies have been conducted to determine whether the physiological influences due to sex hormone differences affect age-related changes in the immune system.
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We also investigated the impact of transient changes in the sex hormone profile at puberty (2 months), and post-puberty (10 months) in intact mice as well as the androgens’ deprivation of castrated mice. Preliminary work (Aboudkhil, 1991) has shown that plasma testosterone concentration in BDF1 4 month-old mice decreases and that it reaches a minimum level at 10 months. The C57BL/6 mice are considered to be sexually and immunologically mature (Rao et al., 1996) at 2–3 months. Compared to the control animals, post-pubertal old mice and castrated mice had fewer CD4+, CD8+ and CD2+ cells in the spleen. Testosterone injections are known (Aboudkhil et al., 1991) to be involved in the distribution of some T lymphocyte subsets, at least in CD4+ and CD8+ thymocytes and splenocytes. As the relative numbers of mature T cells (CD4+ CD3+ and CD8+ CD3+) were decreased in the spleens of castrated animals (Viselli et al., 1995), a reverse effect was observed in testosterone treated mice. The number of CD2+ and CD8+ cells in the spleen increases gradually after birth to reach a plateau at around 3 months of age, and decreases after 12 months of age (Utsuyama and Hirokawa, 1987). Testosterone concentration is high at birth and then decreases (Murad and Haynes, 1985). In our results, there was a correlation between the proportion of CD4+ (r = 0.7, P < 0.05), and CD8+ (r = 0.95, P < 0.01) in the spleen and the plasma testosterone concentration in BDF1 mice. The decrease in spleen T lymphocyte subsets (CD4+, CD8+ and CD2+) and in the blood (CD4+ and CD2+) in 10 month-old and castrated animals could be related to the decrease in the plasma testosterone. Our data suggest that androgens have a physiological effect on the age-related distribution of lymphocyte T subsets. We also examined the influence of physiological (pubertal and post-pubertal age) androgens, castration and DT treatment on the murine leukemia P388 development. The weight and the rate of tumor growth were higher in post-pubertal old and castrated mice than those in young mice. The relative number of CD4+, CD8+ and CD2+ splenocytes in grafted DT untreated mice was low. However, CD8+ and CD4+ splenocytes in the blood of 2 month-old tumor bearing mice were more numerous. The incidence of most types of cancer is higher in aging humans and animals. The high rate of tumor development in old mice can be explained by the immune deficient state associated with aging (Anisinov, 1987). The percentage of CD2+ and CD8+ in the spleen cells of mice with large tumors appeared to be much lower than the percentages in normal mice and mice with a small tumor. The responses of the spleen cells of mice with tumors to three mitogens (PHA, ConA, and LPS) had considerably reduced (Buessow et al., 1984). In fact, in young mice, the decrease of T lymphocyte subsets observed in the spleen was counterbalanced by a
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subset increase (CD4+ and CD8+) in PBL, whereas in 10 month-old and castrated mice, the T lymphocyte subsets of PBL remained unchanged. The lymphocyte cytotoxicity activity of CD8+ cells seems to be important and a crucial effector system for inhibiting tumor growth in vivo (Geissler et al., 1997). As the number of CD8+ cells in the blood of 2 month-old mice is high, it could explain the difficulty for the tumor to develop. DT injection highly decreases the percentage of tumor bearing mice in young, old and castrated ones. Over 30 d, 40% of the old and 60% of the castrated mice died; on the other hand, 16.7% of mortality was found in younger ones. Moreover, testosterone inhibits the in vivo tumorigenic properties of the 1246-3A cells. Castrated male mice receiving injections of 1246-3A cells developed a larger tumor at a higher frequency than sham-operated animals. The administration of testosterone to castrated male mice resulted in a dramatic decrease in tumor development (Serrero et al., 1992). Cross and Dexter (1991) show that testosterone may inhibit tumor growth in vivo by modifying vascularization, which is necessary for tumor development. The decrease in serum level of dehydroepiandrosterone may be associated with patients who have some clinical subtypes of adult T-cell leukemia (ATL) (Kimiharu et al., 1996). Testosterone can inhibit the proliferation and the clonogenic potential of the human monoblastic leukemic cell line U937. The growth inhibition was associated with cell cycle arrest, U937 cells accumulating in G2/M phase (Mossuz et al., 1998). In addition, the inhibition of tumor growth was strongly associated with the level of CTL activity present in CD8 cells derived from the spleen. Moreover, the level of CD8+ CTL activity directly correlated with the degree of inhibition of tumor growth (Serrero et al., 1992). Our results show that CD8+ cells increased in the three batches of animals under DT treatment. A positive correlation was found between the percentage of blood CD8+ cells, CD4+ of spleen cells and tumor weight. Thus, we suggest, besides the direct effect on malignant cells, testosterone can inhibit tumoral growth by modifying CD8+ and CD4+ cells distribution. Recent work (Segal et al., 2002) demonstrated that IL-10 producing CD4+ T cells can manifest anti-tumor functions. The IL-10 maintains the number of anti-tumor CD8+ T cells. Liva and Voskuhl (2001) showed that testosterone can act directly via androgen receptor on CD4+ T lymphocyte to increase IL-10 gene expression. The testosterone action on the growth inhibition may act through CD4+ and CD8+ cells and their cytokine mediators. Our results support other researchers’ propositions (Mossuz, 1998; Blagosklonny and Neckers, 1994; Kimiharu et al., 1996) about the fact that sexual hormones could be used as an adjuvant to improve the chemotherapeutic activity of an anti-tumor agent in leukemia patients.
4. Materials and methods 4.1. Animals BDF1 male mice (C57BL/6 × DBA/2)F1, 3 weeks old, and 10 month-old (IFFA-CREDO, Les Oncins, France), were housed in an air conditioned room (temperature 21 ± 2 °C) in standard cages housing five animals each. Food and water were available ad libitum. The mice were kept on a 12 h light–dark cycle (LD 12:12 h). 4.2. Castration Male mice were castrated at the age of 4 weeks. They were anesthetized with ether and castrated by making an abdominal incision, removing the testes and closing the incision with 6-0 silk sutures. As controls, there were two groups: shamoperated and non-operated (intact) animals. It was found that there were no significant results between the sham-operated group and the non-operated controls; hence the data from these two groups were pooled. The castrated mice were 2 months old, when they were killed. Castration was tested only in 2 month-old mice. The 10 month-old mice were not castrated as their plasma testosterone level was low. 4.3. Steroids Depo-testosterone (DT, Testosterone 17beta-cypionate, SIGMA, St-Louis, MO, USA) in peanut oil (PNO) was administered subcutaneously in mice at a dose of 0.5 mg/100 g body weight, at a final volume of 0.2 ml. The same volume of PNO was injected into the castrated and intact control group. DT was administered to animals on alternate days for a period of 2 weeks. Steroids were injected into treated animals 1 week after castration. The dose, route and frequency of DT administration were chosen based on the work of Ahmed et al. (1985). 4.4. Transplanted mice P388 tumor cell line (from mouse DBA/2 monocyte–macrophage lymphoma) was obtained from Flow, France. They were maintained in culture in Fisher medium supplemented with 5% fetal bovine serum, 1% glutamine (2 mM) and 1% penicillin–streptomycin, at 37 °C in a humidified atmosphere of 5% CO2. The cells were counted with a Coulter counter (ZB, Coulter, Margency, France). Trypan blue exclusion indicated that the viability exceeded 90%. The cells were injected subcutaneously at 105 cells per mouse and the mice were examined daily to monitor survival and tumor growth. Mice were divided into two batches and killed after 15 d or 30 d after tumor grafting. The P388 tumors could only be followed for approximately 2 weeks after which the animals developed leukemia and died (Aabo et al., 1989). Control mice were injected with Fisher medium, or with PNO. No difference was observed between the two batches.
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4.5. Serum testosterone Mice were killed by cardiac exsanguination. The blood was collected in heparinized syringes and the plasma was separated by centrifugation. The testosterone concentration in the plasma was measured by radio-immunoassay by Dr. G. Baudin (Department of Nuclear Medicine, CHU-Nîmes).
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4.9. Statistical analysis The data were analyzed with Student’s t-test and standard correlation. A P value of less than 0.05 was considered significant. The data were plotted using Cricket Graph software.
Acknowledgements 4.6. Cell preparation The thymus and spleen were removed from each mouse and weighed. The weight of the organs was expressed as mg/100 g body weight. The spleen was placed in individual Petri dishes containing phosphate-buffered saline (PBS). Single-cell suspensions were prepared and the cells were washed twice in cold PBS containing 2% bovine serum albumin, 0.01 % sodium azide, and later resuspended in an appropriate volume. Erythrocytes were lyzed by adding 2 ml of lyzing solution to spleen cells and blood samples. The cells were counted in a Coulter counter. The viability was determined by trypan blue exclusion.
4.7. T cell phenotype studies Each sample of lymphoid cells was labeled by incubation with 4 µl monoclonal antibody (MoAb) conjugated to fluorescein (FITC) plus 4 µl MoAb conjugated to phyco-erythrin (PE) for 20 min at 4 °C. The samples were then washed in cold PBS and the cells suspended in 1 ml PBS. The MoAbs used for the phenotypic analysis were thy 1.2-FITC (CD2, clone 30H12, Becton Dickinson, Mountain View, CA, USA), which recognizes immature and mature T cells; Lyt-2-FITC (CD8, Clone 53-6.7, Becton Dickinson) which recognizes cytotoxic/suppressor cells; and L3T4 (CD4, Clone GK1,5, Becton Dickinson) which recognizes helper/inducer cells.
4.8. Flow cytometry analysis The T cell phenotype study was performed using a flow cytometer FACScan (Becton-Dickinson, Mountain View, CA, USA) equipped with an Innova 90-5 (Coherent, Palo alto, CA, USA) argon ion laser operating at 488 nm and 515 mW in light-regulated mode. Light scattering data and fluorescence parameters were collected by user-defined protocols and stored in list mode via Lysis II program. The phenotypic analysis, considering the proportion of positive cells, was determined from histograms, and the red (for L3T4) and green (for Lyt-2 and Thy1,2) fluorescences were used to determine the number of cells per channel based on 20,000 events gated in the lymphocyte area of cytograms: forward angle scatter vs. right angle scatter.
We thank Dr. G. Baudin for the dosage of testosterone in the Department of Nuclear Medicine. This work was supported by a grant from Ligue nationale contre le cancer (comité du gard et de l’aude).
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