Biosci Rep (2006) 26:159–169 DOI 10.1007/s10540-006-9012-1 ORIGINAL PAPER
Synergistic Effect Between Ouabain and Glucocorticoids for the Induction of Thymic Atrophy Sandra Rodrigues-Mascarenhas Æ Neusa Fernandes dos Santos Æ Vivian M. Rumjanek
Published online: 2 June 2006 Springer Science+Business Media, Inc. 2006
Abstract The present report shows that thymocyte death, induced by glucocorticoids, may be modulated in vivo by ouabain. Young, ten days old, mice injected with 140 mg/kg sodium succcinate of hydrocortisone (HC) intraperitonially (i.p.) displayed, 24 h after the injection, a decrease in thymus size and cellular content, an effect that was magnified when ouabain (OUA) 0.56 mg/kg, i.p. was given 1 h prior to the HC injection. Ouabain per se was not capable of producing these changes. Both HC and the combination OUA plus HC induced the death of immature double positive lymphocytes (CD4+CD8+) whereas CD69+ cells survived both treatments. An increase in annexin positive cells and a decrease in mitochondrial membrane potential, assessed by cytofluorimetry, using the fluorescent dye DiOC6, was observed in thymocytes from HC treated animals indicating apoptosis of these cells. Furthermore, a synergistic effect between OUA and HC was also observed using this parameter. The synergy observed in the thymus of animals treated with glucocorticoids and OUA might occur under stress, when both hormones are released, or in situations when ouabain is administered exogenously in a moment of the circadian cycle when glucocorticoid levels are elevated. However the impact of this effect on the immune response is still unknown. Keywords
Atrophy Æ Cell death Æ Glucocorticoid Æ Ouabain Æ Thymocytes
S. Rodrigues-Mascarenhas Æ N. F. dos Santos Æ V. M. Rumjanek (&) Laborato´rio de Imunologia Tumoral, Instituto de Bioquı´mica Me´dica, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro, Bloco H - Sala 003, 21941-590 Rio de Janeiro, RJ, Brazil E-mail:
[email protected] S. Rodrigues-Mascarenhas Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil N. F. dos Santos Center for Structural Molecular Biotechnology (CBME/CEPID/FAPESP), Physics Institute of Sa˜o Carlos, University of Sa˜o Paulo, Sa˜o Paulo, Brazil
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Introduction The thymus is the major site of T lymphocyte development. The early migrants from the bone marrow enter the thymus and undergo a complex program of proliferation, differentiation and cell death where only 5–10% survive to generate mature T cells (Chow et al. 1997; Wilkinson et al. 1995 ; Hare et al. 1998). Thymocyte development requires a continous interaction with the thymic microenvironment and is also influenced by the endocrine and nervous systems. The process of T cell maturation and development occurring within the thymus involves several steps. The progenitor cells originating from the bone marrow and entering the thymus do not express the T cell receptor (TCR) and lack the surface molecules CD4 and CD8, being known as double negative thymocytes (CD4)CD8)). This population differentiates, starts to express both molecules and expands into the CD4+ CD8+ double positive phenotype expressing low levels of TCR. This double positive population represents more than 70% of the thymocytes (Marrack et al. 2000; Kisielow and von Boehmer 1995). At this stage, these cells are the most sensitive to glucocorticoid-induced apoptosis (Chow et al. 1997) and are subjected to positive and negative selection. Interactions between the major histocompatibility complex (MHC) from the thymic epithelium and the TCR complex from the thymocytes are known to be essential to determine whether maturation proceeds to the CD4 or CD8 lineage (Hare et al. 1998; von Boehmer et al. 2003). Positively selected thymocytes up regulate the early activation molecule CD69 and down regulate either CD4 or CD8 from the cell surface to generate mature single positive thymocytes expressing high levels of TCR (Ziegler et al.1994; Swat et al.1993) that will migrate to the periphery. It is known that a number of steroid hormones are capable of reducing thymic volume and promoting thymocyte apoptosis (Pearce et al. 1981). Among these steroids, glucocorticoidinduced thymocyte apoptosis is well established (Nordeen et al. 1976; McConkey et al. 1989; Petit et al. 1995; Mann et al. 2000; Wiegers et al. 2001). Glucocorticoids are known to play a physiological role during thymocyte selection and thymocyte maturation (Vacchio et al. 1998), and a pathological role under stress conditions (Cohen 1992; Olsen et al. 2001; Zacharchuk et al. 1991). Like corticoids, ouabain, previously known as a cardiotonic steroid capable of inhibiting the Na+ K+-ATPase, has recently been identified as a endogenous compound produced by the adrenals and hypothalamus and found circulating in the plasma (Ferrandi et al. 1997; Hamlyn et al. 1991; Laredo et al. 1994, Schoner 2000). Additionally, it has been suggested that ouabain is also released under stress conditions (Goto et al. 1995; Bauer et al. 2005). Work from our group demonstrated that, in vitro, ouabain was capable of inhibiting cell cycle progression of activated mature lymphocytes (Pires et al.1997), inducing apoptosis in these cells (Olej et al. 1998), producing a rapid elevation of intracellular calcium in thymocytes (Echevarria-Lima et al. 2003) and increasing the expression of the early activation molecule CD69 on the thymocyte surface (Rodrigues-Mascarenhas et al. 2003). Furthermore, Mann et al. (2001) observed that the plasma membrane depolarization produced on thymocytes by corticoids might be magnified in the presence of ouabain. These evidences opened the possibility that ouabain might interfere with the maturation process of thymocytes or even produce a synergistic effect with corticoids during pathological stress. The aim of the present work was to verify the in vivo effect produced by ouabain or the combination corticoids plus ouabain on thymocyte maturation.
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Materials and Methods In vivo Treatment with Ouabain and Hydrocortisone Inbred young (10–13 days) Balb/c or C57Bl-6 mice were used in the experiments. Animals were housed in a temperature-controlled room and received water and food ad libitum. During all the experiments performed, animals were treated in accordance with published regulations for animal laboratorial use. In a preliminar experiment 6 groups (2 animals per group) were inoculated i.p. with 2 concentrations of hydrocortisone (70 mg/kg or 140 mg/kg): group 1-control mice treated with 100 ll of complete medium, group 2 – treated with 0.56 mg/kg ouabain (OUA-Sigma, USA) in medium, group 3 – treated with 140 mg/kg sodium succinate of hydrocortisone (HC-Upjohn), group 4 – treated with 70 mg/kg HC, group 5 – treated with 0.56 mg/kg OUA and 1 h later 140 mg/kg HC and group and 6 – treated with 0.56 mg/kg OUA and 1 h later 70 mg/kg HC. In other experiments, four groups of animals were inoculated i.p. with different treatments as above but only the dose of 140 mg/kg HC was used. Two further groups were also used to control the stress produced by the inoculation: group 5 – treated with OUA and inoculated 1 h later with 100 ll of complete medium and group 6 – inoculated with complete medium and 1 h later with 140 mg/kg HC. Twenty-one hours later the thymus of each animal was removed and its weight measured using an analytical balance. Subsequently, thymocytes were isolated and resuspended as described below. Preparation of Thymocytes Suspension Mice were sacrificed by cervical dislocation and their thymi were removed. Cell suspensions were prepared, centrifuged at 300 · g for 7 min and resuspended in RPMI 1640 (Sigma, Chemical Co. USA), supplemented with 5 · 10)5 M b-mercaptoethanol, penicillin 60 mg/l, streptomycin 100 mg/ml, all obtained from Sigma, and 10% foetal calf serum (FCS-HighClone, USA). The thymocyte number was counted under phase contrast microscopy and cell number adjusted to 1 · 106 cells/ml. Flow Cytometry Thymocytes were analysed for CD8, CD4 or CD69 expression after the different treatments. For this, 1 · 106 cells/ml were incubated with a saturating amount of FITC conjugated antimouse CD8 (R&D Systems, USA), PE conjugated anti-mouse CD4 (R&D Systems, USA) or PE conjugated anti-mouse CD69 (Pharmingen, USA) for 30 min at 4C. To prevent nonspecific binding, samples were preincubated for 5 min with normal mouse serum. Cells were then washed twice with chilled PBS and resuspended in PBS. Stained cells were analysed by FACSCalibur (Becton Dickinson, USA). Among harvested cells (10,000 cells/group), viable thymocytes were gated and cell death was evaluated by flow cytometric examination of cells by forward and side-scatter analysis. The gated cells were analysed for the expression of CD4, CD8 and CD69. Data were analysed by WinMid software. Measument of Phosphatidyl Serine Externalization In apoptotic cells, the membrane phospholipid phosphatidyl serine is translocated from the inner leaflet of the plasma membrane to the outer leaflet. To measure phosphatidyl serine externalization an Annexin V-FITC apoptosis detection kit ( BD Biosciences, USA) was used.
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Cells were analysed by FACSCalibur (Becton Dickinson, USA). Among harvested cells (10,000 cells/group) apoptotic cells were identified by positive stain for Annexin V-FITC (region M1). Data were analysed by WinMid software. Measurement of Mitochondrial Membrane Potential using DiOC6(3) To evaluate the mitochondrial membrane potential (DYm), the dye 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)] (Sigma, Chemical Co. USA) was used. The dye was diluted in RPMI and used at a final concentration of 5 nM. Cells were incubated with DiOC6 (3) for 40 min at 37C and 5% CO2.As a positive control, 50 lM of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (Sigma, Chemical Co. USA) was used in the last 20 min of incubation. DiOC6(3) was diluted in ethanol and stored at )20C. Samples were analysed by flow cytometry, detected by FL1 channel and analysed by WinMid software. The region M1 represents cells that have lost the mitochondrial membrane potential. Statistical Analysis Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) followed by the student t test to compare drug-induced changes with respective controls. A value of P < 0.05 was selected as indicating statistical significance and data were expressed as the mean – SD.
Results Effect of OUA Injected In vivo on the Thymic Weight and Cellularity To study the effect of OUA in vivo the doses of 0.56 mg/kg for OUA and 70 or 140 mg/kg for HC in 100 ll were injected i.p. into 9–13 days old mice and the cellularity and weight of thymi, from the animals submitted to different treatments, were measured. A dose dependent effect was observed when HC was used and ouabain magnified the effect of both HC concentrations (Fig. 1 A, B). OUA did not affect the thymic weight when compared to the weight of control animals. HC alone was able to produce a significant atrophy of the thymi. However, treatment with HC in animals that previously received OUA, showed an additional thymic atrophy. To dismiss the possibility that the synergistic effect observed was due, not to OUA, but to corticoid release by the adrenal as a result of stress, another group of animals received culture medium instead of OUA and after 1 h this group of animals received HC (Table 1). It was possible to observe that atrophy depended on the synergistic action between these two drugs. Furthermore, the decrease in thymic weight corresponded to a reduction of cellularity in thymi (Table 1). This same effect could be observed independent of the mouse strain utilized, Balb/c or C57Bl-6. Thymocyte Cell Death Induced by OUA and HC after In vivo Treatment The present paper used a simple and rapid flow cytometric method to verify if the synergistic effect observed, when animals were treated with a combination of OUA and HC, was due to cell death. It was possible to identify cell death according to the appearance of a forward
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A
B
OUA + HC 70mg/kg OUA + HC 140mg/kg HC 70mg/kg HC 140mg/kg OUA Medium 0
10
20
30
40
50
60
Thymus weight (mg)
0
0,5
1
1,5
2
8
Cell Number (x 10 )
Fig. 1 Synergy produced by Ouabain with different doses of Hydrocortisone. Nine- to thirteen-days-old Balb/c mice were injected i.p with the various treatments. When ouabain (OUA) and hydrocortisone (HC) was given to the same animal there was 1 h interval between the injection of OUA and that of HC. Mice were injected 21 h previously with two doses of HC: 70 mg/kg or 140 mg/kg. (A) Thymus weight. (B) Cell number
Table 1 Effect of ouabain (OUA) injected in vivo on the thymic weight and cellularity of animals treated or untreated with hydrocortisone(HC) Treatment Experiment 1 Control (n = 7) OUA (n = 7) OUA + Medium (n = 1) HC (n = 10) Medium + HC (n = 1) OUA + HC (n = 10) Experiment 2 Control (n = 11) OUA (n = 11) OUA + Medium (n = 6) HC (n = 11) Medium + HC (n = 6) OUA + HC (n = 11)
Thymic Weight (mg)
Cell Number (· 108)
51.7 51.0 49.8 31.1 32.3 20.3
– – – – – –
2.9 2.9 0 2.4 0 3.2
1.64 1.74 1.66 0.69 0.52 0.28
– – – – – –
43.1 42.8 43.7 32.6 32.2 19.5
– – – – – –
6.8 6.3 6.8 4.9 5.6 5.4
1.83* 1.60 1.55 0.82 0.78 0.45
0.13 0.16 0 0.14 0 0.10
Nine to thirteen-days-old mice were injected i.p. 21 h previously with: HC 140 mg/kg, OUA 0.56 mg/kg or a combination of both. Thymus weight was measured using an analytical balance. The thymocyte number was counted under a phase contrast microscope. The results are expressed as mean – SD. The groups that received HC and OUA + HC were significantly (P < 0.05) different from the control group that received OUA alone. The groups HC and OUA + HC were significantly different from one another. (*) Cell number represents a pool of thymocytes from all the animals in the group divided by the number of animals
scatter low/side scatter increased population. Corticoids are known to produce thymocytes death and our data demonstrated that HC was capable of decreasing the number of viable cells. Furthermore, when mice were treated with OUA plus HC an increased number of dead cells could be observed (Fig. 2B). The possibility that OUA exacerbated the apoptotic process induced by HC was studied by us using annexin V as a measurement of phosphatidyl serine externalization (apoptosis). As it can be observed the synergistic effect in the percentage of apoptotic cells was also a characteristic of the treatment with OUA and HC (Fig. 2A).
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Fig. 2 Apoptosis induced by Ouabain and Hydrocortisone after in vivo treatment. Nine- to thirteen-days-old Balb/c mice were injected i.p., 21 h previously, with 140 mg/kg HC, 0.56 mg/kg OUA or a combination of both. (A) M1 represents the percentage of apoptotic cells. (B) The numbers represent the percentage of cells according to forward and side scatter parameters. The number on the right hand side represents viable cells. One representative of five experiments is shown
Thymocyte Subpopulations Affected by OUA and HC after In vivo Treatment It is known that immature double positive cells are highly sensitive to HC action. In the present experiments, increased cell death could not be detected when OUA was used in isolation, while HC alone was selective to immature double positive CD4+ CD8+ thymocytes, inducing their death (Table 2). The combination of HC with OUA also led to the death of the double positive population, producing an increase of the double negative subpopulation (Table 2). CD69 Expression on Thymocytes after Treatment with OUA and HC Thymocytes that had been through the process of positive selection up regulate the early activation molecule CD69 and generate mature single positive cells maintaining the expression of CD69 (Anderson et al. 1994). After in vivo treatment, OUA by itself was incapable of Table 2 Effect of ouabain (OUA) and/or hydrocortisone (HC) on thymocyte subpopulations % of cells Treatment
CD4)CD8)
Control OUA HC OUA + HC
3.1 3.4 8.7 15.0
– – – –
0.4 1.6 3.2 5.8
CD4+CD8) 7.3 6.0 26.3 37.5
– – – –
1.9 1.4 11.4 8.6
CD4)CD8+ 2.0 2.3 7.2 8.2
– – – –
0.7 0.8 3.9 1.5
CD4+CD8+ 87.1 86.5 55.0 42.2
– – – –
2.9 2.7 16.1 13.9
Cell suspensions were prepared using thymi from 9 to 13 days old Balb/c mice injected i.p. 21 h previously with 140 mg/kg HC, 0.56 mg/kg OUA or a combination of both. Thymocytes were stained with anti-CD4 and antiCD8 antibodies and subpopulations analysed by flow cytometry. The CD4)CD8) subpopulation from groups HC and OUA + HC were significantly different from one another. Only viable cells were gated and measured based on forward and side scatter
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increasing CD69 expression, whereas HC alone augmented the proportion of CD69 positive cells (Fig. 3). Despite this apparent increase, the absolute number of viable CD69 positive cells indicated that the amount of CD69high cells was unaltered among the various treatments, while the number of CD69low cells was reduced when OUA + HC was given (Fig. 4). These results suggest that there is no increase in CD69 expression but that these cells are more resistant to cell death induced by the various treatments. This effect was evident after the combination of OUA and HC, when no CD69 negative cells remained. Mitochondrial Membrane Potential after OUA and HC Treatment It has been described that the loss of mitochondrial membrane potential is associated to cell death. In the present work we analysed the mitochondrial membrane potential of thymocytes
Fig. 3 CD69 expression on thymocytes after treatment with Ouabain and Hydrocortisone. Nine- to thirteendays-old Balb/c mice were injected i.p., 21 h previously, with 140 mg/kg HC, 0.56 mg/kg OUA or a combination of both. M1 represents auto fluorescence, M2 represents low fluorescence intensity and M3 represents high fluorescence intensity. The numbers represent the percentage of CD69low (M2) and CD69high cells (M3). One representative of five experiments is shown
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Fig. 4 Absolute number of viable CD69 positive cells. Nineto thirteen-days-old Balb/c mice were injected i.p., 21 h previously, with HC 140 mg/kg, OUA 0.56 mg/kg or a combination of both . The figure represents the absolute number of CD69low and CD69high cells. The results are expressed as mean – SD of five experiments
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Number of CD69+ cells (x104)
166 8000
CD69 low
7000
CD69 high
6000 5000 4000 3000 2000 1000 0
Control
OUA
HC
OUA + HC
obtained from animals treated with a combination of OUA and HC. OUA by itself was incapable of decreasing the mitochondrial membrane potential but it was shown to produce a synergistic effect with HC leading to loss of mitochondrial membrane potential (Fig. 5). This is in accordance with the increased number of dead cells observed with this treatment.
Discussion The present report shows, for the first time, that thymocyte death induced by HC may be modulated in vivo by OUA. OUA by itself did not promote cell death, but acts in synergy with HC, producing an increased destruction of this cell type. As described before, thymocytes
Fig. 5 Mitochondrial membrane potential of thymocytes after treatment with Ouabain and Hydrocortisone. Nine- to thirteen-days-old Balb/c mice were injected i.p., 21 h previously, with HC 140 mg/kg, OUA 0.56 mg/ kg or a combination of both. As a positive control 50 lM FCCP was used and added at the same time as the fluorescent dye. The numbers represent the percentage of DiOClow 6 (M1- cells with low mitochondrial membrane high potencial), DiOCint (M3) cells. One representative of four experiments is shown 6 (M2) and DiOC6
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exposed in vivo to HC suffer cell death, leading to a reduction in the number of immature CD4+CD8+ thymocytes. The combination of OUA and HC also led to the death of the same sub-population of immature thymocytes. Glucocorticoid hormones modulate T cell maturation in vivo. While low levels of hormones are required for appropriate T cell development, high levels of glucocorticoid hormones target immature developing CD4+ CD8+ double positive thymocytes for cell death during systemic stress (Wang et al. 1999). The release of ouabain following a stress response (Goto et al. 1995; Bauer et al. 2005) could act in synergy with glucocorticoids and lead to cell death as observed here and, in this way, could influence the development of the immunological repertoire. In the thymus, progenitor T cells are essentially CD4) CD8) CD3). This population expands into the CD4+ CD8+ double positive thymocytes. More than 70% of the cells in the normal thymus express this phenotype. The other two subsets are the mature single positive T cells, CD4+ CD8) CD3+ or CD4) CD8+ CD3+, which have survived the clonal deletion (Cohen 1992; Chow et al. 1997) and express CD69 as a result of positive selection. It is believed that the surface molecule CD69 may be constitutively expressed by some CD4+ CD8+ double positive thymocytes undergoing positive selection and maintained on all mature singlepositive T cells (Anderson et al. 1994). It has been shown that OUA induces the expression of CD69 on the surface of PHAactivated lymphocytes (Pires et al. 1997), and is capable of increasing the levels of CD69 expression on thymocytes after in vitro treatment (Rodrigues-Mascarenhas et al. 2003). However, after in vivo treatment, OUA by itself was incapable of increasing the expression of CD69 on thymocytes. The combination with OUA and HC led to massive cell death and the remaining cells were mostly CD69 positive. It is not clear if the effect produced by OUA is a result of the inhibition of NA+/K+-ATPase or if it involves a different pathway as suggested by Xie (2001). The fact that OUA inhibits the NA+/K+-ATPase could indirectly lead to increased cytosolic Ca2+ levels via Na+/Ca2+ exchange (Balasubramanyam et al. 1994). In accordance with this, dose-dependent intracellular Ca2+ mobilization was observed in thymocytes treated in vitro with OUA (Echevarria-Lima et al. 2003). However, under the same circunstances no plasma membrane depolarization was seen in these thymocytes (Echevarria-Lima et al. 2003). Using, in vitro, a much higher concentration of OUA, Mann et al demonstrated that inhibition of NA+/K+-ATPase by 10 mM OUA depolarizes the plasma membrane of thymocytes and potentiates glucocorticoid-induced depolarization of these cells (Mann et al. 2001). In the present work, we focused on the mitochondrial potential of thymocytes obtained from animals treated with OUA. Although, OUA by itself did not modify mitochondrial membrane potential it produced a synergistic effect with HC leading to loss of mitochondrial membrane potential. This is in accordance with the cell death observed in the thymus and the activation of an apoptotic pathway. Furthermore, other authors have demonstrated that OUA enhances apoptosis of different cells, including peripheral blood lymphocytes (Esteves et al. 2005), cortical neurons (Xiao et al. 2002) and telencephalic cells (Brines and Robbins 1992). The synergistic effect described here and observed in vivo in the thymus of animals treated with glucocorticoids and ouabain might occur under stress, when both hormones are released, or in situations when ouabain is administered exogenously in a moment of the circadian cycle when glucocorticoid levels are still high. The impact of this effect on the immune response is now under evaluation. Acknowledgments This work was supported by the Brazilian National Research Council (CNPq, PRONEX) and FAPERJ (Fundac¸a˜o de Amparo a` Pesquisa do Rio de Janeiro). Sandra Rodrigues-Mascarenhas was a
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recipient of a PhD Fellowship from CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior). We would like to acknowledge Dr. Ottilia R. Affonso-Mitidieri for revision of the text.
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