Murine Model for Human Down's Syndrome )65Dn Mouse, a 16 ...

Reactive Oxygen Intermediates During Programmed Cell Death Induced in the Thymus of the Ts(1716)65Dn Mouse, a Murine Model for Human Down’s Syndrome1 Jesu´s E. Paz-Miguel,* Reyes Flores,† Pablo Sańchez-Velasco,* Gonzalo Ocejo-Vinyals,* Juan Escribano de Diego,* Jacobo Lo´pez de Rego,† and Francisco Leyva-Cobiań2* Down’s syndrome (DS) is one of the most frequent genetic disorders in humans. It has been suggested that overexpression of copper-zinc superoxide dismutase (SOD-1) in DS may be involved in some of the abnormalities observed, mainly neurodegenerative and immunopathological processes. One of the consequences is early thymic involution. Recently, Ts(1716)65Dn mice (Ts65Dn mice), made segmentally trisomic for a chromosome 16 segment, fulfill the criteria for a DS model. To study the possible role of SOD-1 overexpression in thymocyte biology, we analyzed the role of reactive oxygen intermediates during in vivo and in vitro programmed cell death (PCD) induced in the thymus of Ts65Dn mice. Our main findings can be summarized as follows. Ts65Dn thymuses exhibit greater PCD activity than controls, as ascertained by a combination of morphological, histochemical, and ultrastructural procedures. Ts65Dn thymocytes were highly susceptible to PCD induced by both LPS (in vivo) and dexamethasone, a synthetic glucocorticoid agonist (both in vivo and in vitro). Thymus abnormalities were probably caused by SOD-1 hyperexpression in Ts65Dn cells, in that reactive oxygen intermediate generation (specifically H2O2 production) is enhanced in thymocytes and clearly correlates with apoptosis. Similarly, oxidative injury correlated with the formation of lipid peroxidation by-products and antioxidants which partly inhibit PCD in thymocytes. The Journal of Immunology, 1999, 163: 5399 –5410.

D

own’s syndrome (DS)3 is one of the most common human genetic disorders (1, 2). It is caused by the triplication of chromosome 21. Individuals with DS have been reported to have immunological alterations likely to be a result of diminished T cell function. Abnormalities in peripheral blood T cell subsets as well as in their thymus and bone marrow have been observed in DS patients (3). To explore pathogenetic mechanisms in DS, investigators have produced and characterized animal models. Mouse chromosome 16 is the chromosome most homologous to human chromosome 21, and at least 14 defined genes have been localized on both the human and mouse chromosomes in the critical DS region. Mouse fetuses with trisomy 16, a mouse model for human trisomy 21, also exhibit abnormalities similar to those found in DS: a hypoplastic thymus; and a decreased number of hemopoietic progenitor cells in the liver (4). Abnormal development of thymus and spleen cells in trisomy 16 diploid chimeric mice has also been reported. Unfor*Servicio de Inmunologıá, Hospital Universitario “Marque´s de Valdecilla,” Instituto Nacional de la Salud, Santander, Spain; and †Departamento de Biologıá Celular, Facultad de Medicina, Universidad Complutense, Madrid, Spain

tunately, trisomy 16 is incompatible with life and postnatal studies could not be performed. Recently, segmentally trisomic mice, Ts(1716)65Dn (abbreviated Ts65Dn), appear to fulfill the criteria for a DS model. These mice are trisomic for a segment of chromosome 16 that is homologous to human chromosome 21. The segment includes material just proximal to App and extends to Mx (5). Hence, much of the distal end of murine chromosome 16, encoding most of the segment shared with the long arm of human chromosome 21 in the q22 region, is translocated to the centromeric end of murine chromosome 17, forming a small translocation chromosome (6). Ts65Dn mice survive to adulthood. This made this model particularly valuable for an experimental design of extended duration. Although preliminary characterization of Ts65Dn mice has revealed several consistent phenotypic abnormalities, some of which resemble those seen in DS patients (5– 8), no immunological studies of this model have been published. To determine to what extent elevated levels of normal gene products encoded by genes on the triplicate chromosome (mainly superoxide dismutase-1 gen) could affect the functional integrity of the thymus, we conducted a systematic study of these mice.

Received for publication June 21, 1999. Accepted for publication September 7, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Materials and Methods

1

Female trisomic mice ((C57BL/6JEi 3 C3H/HeSnJ)F1-Ts65Dn, abbreviated B6EiC3H-a/A-Ts65Dn) were originally obtained from The Jackson Laboratory, Bar Harbor, ME. Carriers were mated with male B6EiC3H-a/A mice, also from Jackson, to maintain this background. They were bred and maintained in pathogen-free environmental facilities. Sterile trisomic males (6) were used for experimental purposes, whereas trisomic females were used for reproduction. Age- and MHC-matched euploid male progeny were used throughout the study as controls.

This work was supported by Grant DGICYT PB94/1063 and in part by Grant FIS 97/1119 (to F. L.-C.). J.E.P.-M. was supported by a fellowship from the “Marque´s de Valdecilla” Foundation. 2 Address correspondence and reprint requests to Dr. Francisco Leyva-Cobiań, Servicio de Inmunologıá, Hospital Universitario “Marque´s de Valdecilla,” Instituto Nacional de la Salud, 39008-Santander, Spain. E-mail address: [email protected] 3 Abbreviations used in this paper: DS, Down’s syndrome; DCFH-DA, 29,79-dichlorofluorescein diacetate; DEX, dexamethasone; PCD, programmed cell death; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; SOD, superoxide dismutase; DMTU, 1,3-dimethyl-2-thiourea; DDC, diethyldithiocarbamic acid; NAC, N-acetyl-L-cysteine; NGS, normal goat serum; DCF, 29,79-dichlorofluorescein; LPO, lipid peroxides; MDA, malonaldehyde; 4-HNE, 4-hydroxy-2(E)-nonenal; PI, propidium iodide; HPA, hypothalamic-pituitary-adrenal.

Copyright © 1999 by The American Association of Immunologists

Animals

General reagents Salmonella typhimurium LPS was from Difco (Detroit, MI). Ferricytochrome c, bovine superoxide dismutase (SOD, EC 1.15.1.1), colchicine, 0022-1767/99/$02.00

5400 PMA, H2O2, D-mannitol, 1,3-dimethylurea, 1,3-dimethyl-2-thiourea (DMTU), 3,39,49,5,7-pentahydroxyflavone (quercetin), IFN-g, HRP (EC 1.11.1.7), dexamethasone (DEX), 3,39-diaminobenzidine tetrahydrochloride chromogen, diethyldithiocarbamic acid (DDC), N-acetyl-L-cysteine (NAC), 2-mercaptoethylamine (cysteamine), N-propyl gallate, NADPH, phenol red, sulfanilamide, naphthylethylene diamine dihydrochloride, SDS, and phosphoric acid were from Sigma (St. Louis, MO). PE and 29,79dichlorofluorescein diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR). Hydrazine carboximidamide hemisulfate (aminoguanidine) was from Calbiochem (San Diego, CA). Biotin-16-29-deoxyUTP (biotin-dUTP), dATP, TdT, and TdT buffer were purchased from Boehringer Mannheim Biochemicals (Mannheim, Germany). Culture media were RPMI 1640 (R0) and DMEM (D0), supplemented with 2 mM D-glutamine and 10% heat-inactivated FCS (HyClone, Logan, UT) (R10 and D10, respectively). Normal mouse, goat (NGS), and rabbit sera were used for blocking. All media and reagents used were confirmed to be endotoxin free (,0.01 ng/ml) by chromogenic Limulus amebocyte lysate microassay from Whittaker M.A. Bioproducts (Walkersville, MD).

Antibodies FITC-labeled rat anti-mouse CD4 (IgG2b, GK1.5), PE-labeled rat antimouse CD8 (IgG2a, 3B5) and FITC-labeled hamster anti-mouse CD3 (IgG, S4.1) mAbs were purchased from Caltag Laboratories (San Francisco, CA). The following Abs were obtained from PharMingen (San Diego, CA): biotinylated rat anti-mouse CD35 (IgG2a, 8C12); biotinylated mouse anti-murine Iak (IgG2b, 11-5.2); biotinylated mouse anti-murine Iab (IgG2a, KH74); biotinylated mouse anti-murine Iad (IgG3, 39-10-8); PErat anti-mouse CD45R/B220(IgG2a, RA3-6B2); biotinylated rat antimouse CD119 (IgG2a, GR20); and purified hamster anti-murine Bcl-2 (IgG, 3F11). Alkaline phosphatase-conjugated goat antiserum to mouse IgG and IgM were obtained from Sigma. Biotinylated F(ab9)2 goat antihamster IgG (Jackson ImmunoResearch, West Grove, PA), streptavidinPE, and streptavidin-Red 670 (Tago, Burlingame, CA) were also used as secondary reagents.

Mitotic chromosome preparations Whole blood from the retroorbital sinus was collected in heparinized microhematocrit tubes. Blood (100 ml) was cultured at 37°C in R10 in the presence of 50 mg/ml LPS and 6 mg/ml PHA for 42 h (9). One-half hour before the end of the incubation time, 0.1 ml colchicine (50 mg/ml) was added to each tube. After centrifugation (250 3 g, 22°C, 10 min) 2–3 ml of a prewarmed 0.56% KCl solution were added to each tube and incubated for 15 min. They were centrifuged again (480 3 g, 22°C, 10 min) and finally fixed (22°C, 30 min) with 3 ml methanol-glacial acetic acid (3:1). Samples were washed three times in freshly prepared fixative before smears were prepared by gentle dropping onto clean slides.

In vivo LPS and DEX administration In preliminary protocols, mice were injected i.p. with various concentrations of LPS, but the data reported here were from mice injected with 50 mg LPS. Three or four animals were used for each experimental group. A similar procedure was adopted for DEX administration. In this case, 2 mg of the drug were injected i.p. into each mouse. The LPS- and DEX-injected animals were sacrificed after 10 or 48 h, respectively. For some in vivo experiments, parallel sets of both control and trisomic mice were pretreated with NAC or DDC (100 mg/kg body weight i.p. injected daily for 2 weeks) before DEX administration. As controls, both euploid and trisomic mice were injected with endotoxin-free sterile PBS in parallel.

Microscopy and ultrastructural studies Thymuses were fixed in Bouin’s fixative for 5 h, rinsed, and transferred into 70% ethanol. Tissues were then processed through alcohols and xylene, embedded in paraffin, sectioned at 5 mm, and either stained with hematoxylin and eosin or used for immunohistochemistry. For in situ thymocyte apoptosis detection, 5-mm-thick sections from fixed, paraffin-embedded tissues were processed for 39-hydroxy-DNA end staining. Cells showing nuclear DNA fragmentation were identified by the TUNEL method (10). Tissue sections were counterstained with methylene green. Samples for electron microscopy were fixed in 4% glutaraldehyde in Sorensen’s buffer and processed routinely.

SOD-1 activity To measure SOD-1 activity, supernatants of tissue homogenates (thymus, erythrocytes, and liver) were collected and assayed with a commercial kit (Calbiochem), according to the manufacturer’s instructions.

THYMOCYTE APOPTOSIS IN Ts65Dn MICE Cell culture Murine thymuses were placed in sterile PBS, and the tissue was disrupted with the tip of the plunger of a sterile 1-ml syringe. Cells were washed in cold PBS and centrifuged three times (400 3 g, 5 min, 4°C) to remove cellular aggregates. Single-cell preparations were counted and viability assayed by trypan blue exclusion. For in vitro experimental procedures, a single-cell suspension (1 3 106/ml) was prepared and cultured with or without LPS (50 mg/ml) or DEX (1 mM) for different periods of time. For some experimental procedures, thymocytes were cultured in the presence of selected scavengers or inhibitors as described. Peritoneal macrophages were obtained as previously described (11) and cultured in appropriate medium for functional studies.

Quantitation of reactive oxygen intermediates (ROI) and determination of nitrite production To quantitate ROI, O22 and H2O2 production were measured. O22 production was measured by the SOD-inhibitable reduction of ferricytochrome c adapted to a microplate format as previously described (12). Results are expressed as nanomols O22 produced per mg protein. Each result represents the mean of eight determinations. H2O2 production was measured by a technique based on the HRPO-dependent conversion of phenol red by H2O2 into a compound with increased absorbance at 600 nm (12). Results are expressed as nanomols H2O2 produced per mg protein. Each result represents the mean of five determinations. In addition, a highly sensitive flow cytometric method was used to detect intracellular H2O2 formation in thymocytes (13); the method used the H2O2-sensitive fluorescent probe DCFH-DA. Thymocytes were twice washed in PBS, resuspended at 1 3 106 cells/ml in phenol red-free HEPES-buffered D10, and incubated with 5 mM DCFH-DA at 37°C. Interaction of DCFH-DA with peroxides gives rise to the fluorescent DCFH. This oxidation reaction leads to the formation of the highly fluorescent molecule 29,79-dichlorofluorescein (DCF) detectable by FACS analysis (14). Approximately 104 cells per time point were analyzed (excitation and emission settings were 488 6 15 and 535 6 15 nm, respectively). Mean fluorescence peak height was determined during 60 min incubation at 37°C. The generation of nitrite (NO22) was used to estimate reactive nitrogen intermediates (RNI) indirectly. It was determined by the microplate method of Ding et al. (12, 15). Results of unknown culture fluids were expressed as nanomols NO22 produced per well; derived from a sodium nitrite standard curve.

Determination of lipid peroxidation by-products Total lipid peroxides (LPO), malonaldehyde (MDA), and 4-hydroxy-2(E)nonenal (4-HNE) were quantitated as described (16). Briefly, cells were cultured at 1–2 3 106 cells/ml for 1 h with or without the reagents to be tested in R0 medium supplemented with 5% FCS, in the absence of phenol red. For LPO determination, cells were washed twice and solubilized with 0.1% Triton-X and 0.05% deoxycholate in saline. For the detection of MDA and 4-HNE, the cells were resuspended in 20 mM Tris-HCl buffer, pH 7.4, and submitted to three cycles of freeze-thawing and further centrifugation. Commercially available colorimetric assays were used to detect LPO (Kamiya Biomedical, Thousand Oaks, CA) and MDA and 4-HNE (British Biotechnology Products, Abingdon, U.K.) according to the manufacturers’ recommendations.

Flow cytometric analysis Two- and three-color flow cytometric analyses of single-cell suspensions of thymus were performed with anti-CD4-, anti-CD8-, anti-CD3-, antiCD45R/B220-, and anti-CD119-conjugated mAbs. In other cases, twocolor cytometry was performed with conjugated anti-CD3, unlabeled hamster IgG anti-murine Bcl-2 followed by staining with biotinylated-goat antihamster and streptavidin-PE, as previously described (17). Cells were analyzed with a FACScan flow cytometer and a minimum of 3 3 104 events per sample were counted using Lysis software (Becton Dickinson, Mountain View, CA). The apoptosis levels in thymocytes were assessed by propidium iodide (PI) staining as previously described (18). Briefly, a pellet of 5 3 105 cells was resuspended in 600 ml H2O containing PI (50 mg/ml), 0.1% sodium citrate, and 0.1% Triton X-100 and then incubated overnight in the dark at 4°C. Cells were analyzed by flow cytometry. To characterize the phenotype of the apoptotic cells, the detection of a given cell surface marker in TUNEL-positive cells was performed by three-color flow cytometry as described previously (19). Briefly, thymocytes were incubated (30 min, 4°C) with the appropriate anti-CD4 and anti-CD8 Abs in 100 ml PBS. After two washings with PBS, they were fixed with 2% paraformaldehyde for 30 min, washed twice again, and incubated for 2 min with 1% Triton X-100 containing 0.1% sodium citrate. After an additional washing step, the TUNEL reaction was conducted by incubating the cells

The Journal of Immunology

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FIGURE 1. Histological characterization of the superficial cortex of normal and Ts65Dn thymus. A, Typical histological pattern of thymus capsule and cortex in normal mice. B, Lymphocyte depletion at the subcapsular level (p) with basophilic apoptotic-like nuclei grouped at this level (arrow) in Ts65Dn thymus. C, Higher magnification of one of these clusters of degenerated lymphocytes. The epithelial cell nuclei are clearly seen. D and E, TUNEL-positive cells in control and Ts65Dn cortical of the thymus, respectively. Original magnifications: A and B, 3350; C, 31000; D and E, 3250. FIGURE 2. Histological characterization of the deep cortex of normal and Ts65Dn thymus. A, TUNEL-positive cells are closely located to the cortical septi in normal thymus. B, TUNEL-positive cells are more abundant in Ts65Dn thymic cortex. C, Hyperchromatic nuclear fragments (arrow) clustered in close vicinity to the deep cortical septi. Original magnifications: A and B, 3250; C, 3350. FIGURE 3. Histological characterization of the thymic medulla of normal and Ts65Dn thymus. A, Sparsely distributed TUNEL-positive cells in a normal thymus. B, TUNEL-positive cells are significantly increased in the Ts65Dn thymic medulla. C, Hassall’s corpuscles (p) with hyperchromatic lymphocytes in the neighboring areas (arrow) in Ts65Dn thymus. Original magnifications: A and B, 3250; C, 3350.

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THYMOCYTE APOPTOSIS IN Ts65Dn MICE

FIGURE 4. Ultrastructural findings in the superficial cortex of normal and Ts65Dn thymuses. A, Subcapsular area in a normal thymus showing small and medium sized lymphocytes with normal proportions of mitosis (m) and apoptosis (a) in some of them. Some macrophages with engulfed materials (arrow). B, Subcapsular cortex in the thymus of Ts65Dn mice. A large number of macrophages with increased phagocytic activity (p) are surrounded by reticular epithelial cell processes (arrow) and numerous apoptotic lymphocytes (a). Original magnifications: A, 32500; B, 33500. FIGURE 5. Ultrastructural findings in the deep cortex of normal and Ts65Dn thymuses. A, Normal cellular density as well as the proportion of apoptotic lymphocytes in this location in control thymuses. A macrophage with remains of phagocytic vacuoles (arrow). B, Cell to cell contact missing with subsequent enlargement of the intercellular space (p) in a similar area in Ts65Dn thymus. A macrophage with numerous lipid bodies in the cytoplasm (arrow). C and D, Different patterns of epithelial cell-lymphocyte relationship in control and Ts65Dn thymuses, respectively. In normal thymus, lymphocytes and epithelial cells adopt an uniform packed pattern without evident intercellular spaces. In Ts65Dn thymus, the cytoplasm is retracted having lost cell contact. The intercellular space is enlarged (p). E, In the vicinity of the cortical septi, epithelial cells and scarce mitotic (m) and apoptotic (a) cells are observed in normal thymus. F, In the same location of Ts65Dn thymus, epithelial cells show signs of vacuolization (p) and a higher proportion of apoptotic cells (a). Original magnifications: A, 32500; B, E, and F, 33000; C and D, 33500.


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Table I. Phenotypic analyses of thymic cell subpopulations in untreated Ts65Dn and control mice and after LPS or DEX treatment in vivoa Phenotype (%) Agent Injected

PBS PBS LPS (50 mg) DEX (1 mg)

Mice

Euploid Ts65Dn Euploid Ts65Dn Euploid Ts65Dn Euploid Ts65Dn

Age (wk)

5–7 12–15 12–15 12–15

1

CD4 CD8

1

60 6 1 49 6 2† 81 6 4 63 6 5† 78 6 2 47 6 8* 80 6 5 59 6 4†

1

CD4 CD8

24 6 3 35 6 2† 10 6 3 22 6 8† 16 6 2 28 6 3† 11 6 4 23 6 7†

2

CD42CD81

CD42CD82

0.9 6 0.1 0.6 6 0.1 2.3 6 0.5 3.1 6 0.4 2.1 6 0.8 4.1 6 0.9† 3.3 6 0.7 4.1 6 1

15 6 1 14 6 1 7.1 6 1.4 11 6 2 4.1 6 2 18 6 3† 6.1 6 1 13 6 3†

a Results were obtained by two-color flow cytometric analysis of thymocytes simultaneously labeled with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 Abs. Values are expressed as the mean 6 SD percentage of stained cells from seven mice per group. p, p , 0.001; †, p , 0.01.

(1 h, 37°C) with 0.3 nmol biotin-dUTP, 3 nmol dATP, 25 U TdT, and TdT buffer. Cells were washed and incubated with streptavidin-Red 670.

DNA fragmentation DNA extraction was performed as described (20). In brief, 2 3 106 thymocytes were lysed in 0.5 ml 10 mM Tris-HCl, pH 8, containing 10 mM EDTA and 0.6% SDS. The DNA was then extracted by phenol-chloroform and ethanol precipitated. Samples were electrophoresed in 2% agarose gel.

Results Ts65Dn mice have enhanced apoptosis of the thymus In both human and previous experimental murine models of DS, the involutive morphological changes and functional abnormalities of the thymus have been described (21–27). Thymic morphology of Ts65Dn mice was compared with normal littermates at 2, 4 and 7–12 wk of age. No remarkable ultrastructural abnormalities were observed in 2-wk-old Ts65Dn mice (not shown). At 4 wk, trisomic mice exhibited slight abnormalities in both cortical and medullar zones, mostly consisting of increased numbers of TUNEL-positive cells, modified epithelial cells, and macrophages (not shown). Changes in thymic morphology of Ts65Dn mice were clearly seen when compared with age-matched normal littermates. TheTs65Dn thymic cortex was remarkably different. At 7–12 wk of age, the lower number of subcapsular lymphocytes (p) with hyperchromatic apoptotic-like clusters (arrows) was evident in Ts65Dn thymuses (see Fig. 1B vs normal thymus in Fig. 1A). At higher magnification (Fig. 1C), rosette-like grouped cells (most probably representing degenerative nuclei) exhibit highly basophilic nuclei of variable size (arrow), in the subcapsular zone. In Ts65Dn mice, numerous grouped TUNEL-positive nuclei in small nests close to epithelial cells were seen (Fig. 1E). The number of TUNEL-positive were specially abundant in areas close to the connective tissue of the capsular and cortical septi of Ts65Dn thymuses (Fig. 2B). This moth-eaten or empty aspect of the Ts65Dn thymic cortex could be observed through all their thickness but was more remarkable in areas close to the connective septi (Fig. 2C, arrows). Grouped TUNEL-positive cells appeared more frequently in Ts65Dn thymus than in control thymic medulla at the level of both Hassall’s corpuscles and vessels (Fig. 3B). Isolated hyperchromatic nuclei (arrows) could also be seen homogenously distributed in the vicinity of Hassall’s corpuscles (p) and perivascular spaces (Fig. 3C). In conclusion, the majority of apoptotic thymocytes in

situ were detected at the corticomedullary junction, although they were also found scattered throughout the cortex. The ultrastructural morphology of the cellular organization showed changes and with evident signs of hypocellularity. Grouped hyperchromatic nuclei correspond to apoptotic lymphocytes (retracted hyperchromatic nuclei and degenerative signs in the cytoplasm) and macrophages with evidence of active phagocytosis (Fig. 4B vs normal thymus in Fig. 4A). In the bosom of large and empty spaces, epithelial cells showed numerous filiform cytoplasmic processes, and there were few contacts with normal lymphocytes (Fig. 4B). In the Ts65Dn thymus cortex, macrophages exhibited high content in lipidic bodies (Fig. 5B). The loss of cellular contact between both cell populations became more evident in the deep cortex of Ts65Dn mice, where epithelial cells show irregular cytoplasm with dendritic processes and no contacts with neighboring floating lymphocytes (Fig. 5D). The nuclear membrane as well as the saccules of the endoplasmic reticulum of these rounded and vacuolated epithelial cells was abnormally enlarged (Fig. 5F vs normal thymus in Fig. 5E). No dramatic alterations in the microvasculature at the level of the corticomedullar junction and the medulla itself were observed. However, cellular retraction, a certain degree of intracellular edema, and residual apoptotic cells were frequently seen in similar areas of the trisomic thymus (Fig. 6B). At the level of the Ts65Dn thymic medulla, the stromal populations showed little activity, with epithelial cells exhibiting large numbers of tonofilaments in their prolongations, macrophages had residual phagocytosed bodies and numerous apoptotic lymphocytes (Fig. 6D). When Ts65Dn mice were treated in vivo with apoptotic drugs (LPS or DEX), these changes increased accordingly as were expected (not shown). Similar observations were obtained in LPStreated mice made transgenic for human SOD-1 (28, 29). Older animals (.20 wk of age) were not evaluated because of the thymic involution observed in aged mice. Phenotypic analysis of thymocytes was performed by two-color flow cytometry using cells labeled simultaneously with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 Abs (Table I). These studies were performed in thymuses from Ts65Dn mice as well as in control littermates of different ages. In general, the proportion of CD41CD81 subpopulation was clearly diminished in trisomic mice at both 5–7 and 12–15 wk of age. By three-color

FIGURE 6. Ultrastructural findings in the thymic medulla of normal and Ts65Dn thymuses. A, Normal endothelium and perivascular space with few signs of apoptosis (a). B, The vessels show slight edematous intercellular spaces and high apoptotic activity (a) in Ts65Dn thymus. C, Clustered epithelial cells with discrete vacuolization (p) in normal thymus. D, Increased number of tonofilaments (arrow) and residual bodies (p) in the cytoplasm of epithelial cells and macrophages, respectively, in the Ts65Dn thymuses. Original magnifications: A, 32000; B, 32500; C and D, 33000.

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THYMOCYTE APOPTOSIS IN Ts65Dn MICE

FIGURE 7. Apoptosis in thymocytes of control (Eu) and Ts65Dn (Ts) mice after in vivo injection of LPS and DEX. Thymuses from 8-wk-old Ts65Dn and control mice were removed 12 and 24 h after i.p. injection of LPS (50 mg) and DEX (1 mg), respectively. Then, thymuses were teased apart, cells collected, and cultured for up to 24 h. Left, Cell viability in thymic cells from LPS-injected mice and DEX-injected mice. Right, DNA fragmentation after LPS injection. DNA from thymocytes of control and Ts65Dn mice was extracted 12 h after i.p. injection of PBS or LPS (10 and 50 mg), and the intensity of DNA fragmentation associated with apoptosis was analyzed.

cytometry, the larger population of apoptotic thymocytes corresponded to the CD41CD81 phenotype (see below and Fig. 9). Ts65Dn thymocytes exhibited accelerated cell death after administration in vivo of apoptotic-inducing agents It is well known that both Gram-negative and Gram-positive bacteria induce thymic atrophy via apoptosis in mice (30, 31). LPS of Gram-negative bacteria has been reported to be a major factor in the pathogenesis of Gram-negative septic shock (32). In vivo injection of LPS also induces loss of thymic weight and a decrease in thymic lymphocytes (33). On the other hand, it is also well established that immature murine thymocytes undergo apoptosis in response to DEX (34). Specifically, apoptosis in both control and trisomic thymocytes was evaluated after i.p. injection of LPS and the synthetic glucocorticoid agonist, DEX. Thymuses from 8-wk-old control mice were removed 12 and 24 h after i.p. injection of various doses of LPS (0 –50 mg) or DEX (0 –2 mg). Thymic cells were collected and cultured for up to 24 h. Cell viability was recorded, and the appearance of fragmented DNA was examined by gel electrophoresis. There was a dosedependent increase in fragmented DNA by injection with more than 20 mg LPS or 500 mg DEX. Maximal DNA fragmentation was obtained with mice injected with 50 mg and 2 mg LPS and DEX, respectively (these amounts were chosen for additional experiments). A typical experiment is shown in Fig. 7. Both agents accelerated the induction of apoptosis in thymuses of both euploid and trisomic mice, but this effect was, as expected, specially significant in the latter. Apoptosis was demonstrated by counting viable thymocytes (Fig. 7, left) as well as by visualization of fragmented DNA (Fig. 7, right). Efforts were made to determine the phenotype of the thymocytes treated or not with these agents (see below and Table I). Thus, thymocytes from both control and Ts65Dn mice were phenotypi-

cally evaluated by flow cytometry. As is described below, the apoptotic subset (TUNEL1 cells) was that corresponding to the CD41CD81 phenotype in both populations. Thymocytes of Ts65Dn mice are more susceptible to DEX-induced PCD in vitro Glucocorticoid hormones cause rapid depletion of thymocytes by apoptosis in vivo as well as in vitro (34). However, LPS is a wellestablished PCD inducer agent in vivo only (33). Because glucocorticoid and bacterial products appear to induce thymocyte PCD by different pathways, the sensitivity to DEX- and LPS-induced cell death was evaluated in thymocyte cultures. To evaluate thymocyte apoptosis sensitivity of Ts65Dn mice to glucocorticoid-induced and LPS-induced DNA fragmentation, thymocytes were treated with DEX (1 mM) or LPS (50 mg/ml) in culture for up to 18 h. In Fig. 8 (left), the results of a typical experiment are shown. Untreated thymocytes from Ts65Dn showed a higher proportion of apoptotic cells. DEX increased the percentage of apoptotic cells in both thymocyte populations, being significantly higher in Ts65Dn cells, indicating an enhanced sensitivity of these trisomic cells to undergo DEX-driven PCD (Fig. 8, lower left) Flow cytometric analysis revealed that DEX-treated thymocytes displayed a sub-G0 DNA peak, which is known to correspond to fragmented DNA in thymocytes (35). As expected, no significant differences were observed between LPS-treated and untreated thymocytes from both mice (Fig. 8, upper left). In Fig. 8 (right), the DNA fragmentation of both DEX-treated and untreated cells is illustrated. To evaluate the phenotype of the DEX-triggered apoptotic thymocytes, thymic cells obtained from Ts65Dn and euploid littermates and cultured in presence or absence of DEX (1 mM) were stained for simultaneous three-color visualization of CD4 and CD8


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FIGURE 8. Apoptosis in thymocytes of control and Ts65Dn mice after in vitro stimulation with LPS and DEX. Upper and lower left, 1 3 106 thymocytes from 10-wk-old Ts65Dn and euploid mice were cultured in the presence or absence of LPS (50 mg/ml) or DEX (1 mM), for up to 18 h (DEX) or 24 h (LPS). At the end of each incubation period, cells were fixed with 1% paraformaldehyde, permeabilized with Triton X-100, and assessed for nuclear DNA content by PI staining. Cells were analyzed by flow cytometry. Data represent mean 6 SD of apoptotic cells in triplicate cultures for three independent experiments. Right, DNA fragmentation in control and Ts65Dn thymocytes was studied after adding DEX (1 mM) to the cultured cells for 6 h.

combined with an improved technique for TUNEL staining (Red670-dUTP staining, see Materials and Methods) (Fig. 9). Again, quantitative analysis showed that the subset of TUNEL-positive cells corresponded to those stained simultaneously for both CD4 and CD8 molecules. Bcl-2 expression in thymocytes An important factor in shaping the TCR repertoire during thymocyte development is the susceptibility of double-positive (CD41CD81) thymocytes to enter in a program of cell death (negative selection) when the TCR is engaged by self-Ags. Recent evidence has suggested that this susceptibility to PCD might be influenced by the expression of Bcl-2 (see Ref. 36 for a review). For this reason, we considered whether or not Ts65Dn thymocyte cell death could be related to Bcl-2 expression. Approximately 28% of normal thymocytes expressed Bcl-2. A similar percentage occurred in thymocytes from Ts65Dn mice. Bcl-2 was expressed by nearly all CD41CD82 and CD42CD81 subpopulations in both control and Ts65Dn mice, but by only 5–7% of CD41CD81 thymocytes in both mice. In the CD42CD82 subpopulation, 60 – 65% of thymocytes were Bcl-21 (not shown). Moreover, Bcl-2 was found to be expressed throughout the thymic medulla, but only by scattered cells in the thymic cortex (not shown). SOD-1 levels and generation and kinetics of ROI generation by Ts65Dn thymocytes Ts65Dn mice overexpress the homologues of multiple genes located on human chromosome 21 (5, 6). We have measured SOD-1 protein levels in several tissues of Ts65Dn mice. As expected, SOD-1 sp. act. in thymus of trisomic mices was ;1.8 times control values (2.7 6 0.3 vs 1.48 6 0.3 SOD525 U/ml in Ts65Dn and euploid mice, respectively).

The high SOD-1 levels in tissues of Ts65Dn mice may lead to increased production of H2O2 and thus to oxidative stress; and a higher susceptibility of the Ts65Dn mouse thymocytes to PCD. As a preliminary approach we correlated SOD-1 levels with ROI and RNI production in activated macrophages (in these cells oxygen consumption can be easily measured). The steady-state level of oxygen consumption depends on the level of NADPH oxidoreductase activity and on the rate at which O22 and H2O2 are utilized in the cell. Thus, an increased intracellular amount of SOD-1 in cells dismutates a higher amount of O22 to O2. This was observed in macrophages from trisomic mice, which released 4 times less O22 than control cells (7 6 2 vs 30 6 4 nmol/mg in Ts65Dn and euploid cells, respectively). It would be reasonable to expect that overexpression of SOD-1 would also lead to the accumulation of higher concentrations of H2O2. Indeed, Ts65Dn macrophages released 3 times more H2O2 than macrophages from control littermates (94 6 14 vs 29 6 6 nmol/mg in Ts65Dn and euploid cells, respectively). RNI production was also evaluated in macrophages. There was no significant difference in terms of NO22 production up to 1 mg/ml LPS (8.7 6 1 vs 10 6 1 nmol/105 cells in euploid and Ts65Dn cells, respectively). To examine whether the production of intracellular H2O2 is increased in thymocytes from Ts65Dn mice, the level of intracellular peroxides in the thymocytes was determined. This was studied in vivo in thymocytes from mice injected or not with LPS or DEX and in vitro by culturing thymocytes with these apoptosis-triggering agents. ROI production is difficult to measure in thymocytes by conventional procedures. For this reason, intracellular H2O2 levels were assessed using the sensitive DCFH-DA probe (13): Cells from Ts65Dn and control mice before and after in vivo injection of LPS (50 mg) or DEX (1 mg) were treated with DCFH-DA at

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THYMOCYTE APOPTOSIS IN Ts65Dn MICE different periods, and fluorescent DCF was analyzed by flow cytometry as described in Materials and Methods. Fluorescence values of thymocytes from PBS-injected Ts65Dn mice were always higher than in control euploid cells, although these differences were not significant (Fig. 10). On the other hand, statistically significant differences were observed in the fluorescence values between control and Ts65Dn thymocytes from animals injected with LPS (Fig. 10A). Similar results were obtained when H2O2 generation was recorded in thymocytes from DEX-injected mice (Fig. 10C). or when quantitated in thymocytes treated with DEX in vitro (Fig. 10D). No differences in H2O2 production were recorded between Ts65Dn and euploid thymocytes, neither when cultured in medium alone nor after LPS addition (Fig. 10B). These results, together with those of susceptibility to apoptosis, indicate that: 1) thymocytes from Ts65Dn mice produce higher levels of peroxides in comparison with euploid mice; 2) this difference is enhanced by treatment with DEX both in vivo and in vitro; 3) LPS only increase this effect in vivo; and 4) these observations matched very well with susceptibility to apoptosis by these agents in vivo and in vitro.

LPS- and DEX-induced apoptosis is associated with an increased lipid peroxidation in thymocytes from Ts65Dn mice

FIGURE 9. Immunocytometric characterization of thymic apoptotic cells after treatment with DEX in vitro. Approximately 1 3 106 thymocytes obtained from 8-wk-old Ts65Dn and euploid littermate mice were cultured in the presence or absence of DEX (1 mM), for 6 h. Cells were then stained for simultaneous three-color staining of CD4 and CD8 combined with an improved technique for TUNEL staining as described in Materials and Methods. Top, FACS analysis distribution of apoptotic (Red-670-dUTPpositive) cells in subpopulations of thymic cells. Bottom, Quantitative analysis shows the percentage of CD41CD81TUNEL1 cells from both mice treated or untreated with DEX as above.

FIGURE 10. Kinetics of ROI generation by thymus cells. H2O2 production by thymocytes from Ts65Dn and euploid mice was studied after i.p. injection of LPS (50 mg) (A) or DEX (1 mg) (C) in vivo. Similarly, thymocytes from untreated mice were incubated with LPS (50 mg/ ml) (B) or DEX (1 mM) (D) in vitro. In both experimental procedures, control mice or control cultures were injected with PBS or left untreated (medium), respectively. Thymocytes were cultured at 37°C for up to 60 min in the presence of DCFH-DA (5 mM). At different time periods, fluorescent DCF was analyzed in at least 104 cells in a flow cytometer. Results are expressed in arbitrary units of mean fluorescence intensity (MFI).

Lipid peroxidation is an autocatalytic free radical chain reaction stimulated by the highly reactive hydroxyl radical zOH (37). Certain membrane-associated structures can be considered targets of z OH-induced peroxidative damage: mitochondria, cytoplasmic membranes and lysosomes (for a review, see Ref. 37). To correlate apoptosis by LPS and DEX with the effects of these substances on the oxidative burst, we quantitated total lipid peroxides produced by cells treated or not with these apoptosis-inducing reagents. Results of two experiments performed indicated: first, that treatment with increasing amounts of DEX generates detectable and significant levels of peroxides in a dose-dependent manner in thymocyte extracts, whereas untreated cells do not form lipid peroxides (Table II). In addition, because of several problems and the lack of


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Table II. Quantitation of lipid peroxidation by-products in thymocytes cultured in the presence of LPS and DEXa Lipid Peroxides (mmol/L) Incubated with

Medium only LPS (50 mg/ml) DEX (1 mM)

Cells

Total

4-HNE

MDA

Euploid Ts65Dn Euploid Ts65Dn Euploid Ts65Dn

1.8 2.5 ND 3.8 12.9 20.8

,0.3 ,0.3 ,0.3 0.9 4.25 6.1

,0.3 0.8 ,0.3 2.6 5.01 8.01

a Thymocytes were cultured at 1 3 106 cells/ml with the indicated reagents for 1 h at 37°C. Total LPO, MDA, and 4-HNE were determined in samples prepared as described in Materials and Methods. Results are expressed as the mean micromole/ liter of two experiments performed in triplicate.

specificity of many available methods for detecting lipid peroxidation (discussed in Ref. 38), 4-HNE and MDA, two major products of v6-polyunsaturated fatty acids, were also measured (Table II). Almost identical results were obtained. Production of 4-HNE and MDA followed a pattern similar to that of total LPO and was DEX induced. This effect, although to a lesser extent, was also observed with LPS. MDA and 4-HNE are only two of a great number of carbonyl compounds formed and often represent only a small percentage of the total products formed, so quantitation of total lipid peroxides largely exceeded the quantitated 4-HNE and MDA together. Antioxidants inhibit the thymic apoptosis According to the results generated, oxidative stress probably contributed to enhanced apoptosis in the thymus of Ts65Dn mice. To further study the contribution of both ROI and RNI generation in thymocyte apoptosis, Ts65Dn and control thymocytes, treated or not with DEX, were cultured in the presence or absence of a large array of scavengers and inhibitors and dead cell counted as an estimate of apoptosis (Fig. 11). Radical scavengers can be used to abolish intracellular redox processes in intact cell preparations or

FIGURE 11. Reversibility of DEX-induced apoptosis in thymocytes by antioxidants. Different sets of thymocytes from euploid (left upper panel) and Ts65Dn (right upper panel) mice were cultured in parallel with or without 1 mM DEX in the presence or absence of antioxidants for 6 h. Concentrations tested were: aminoguanidine (AG, 20 mM); catalase (100 mg/ml); cysteamine (1 mM); DDC (5 mM); 1,3-dimethylurea (DMU, 100 mM); DMTU (100 mM); mannitol (20 mM); NAC (1 mM); N-propyl gallate (NPG, 5 mM); quercetin (50 mM); SOD (250 mg/ ml); and vitamin E (vit E, 250 mM). Lower panels, dose-response effect of DDC and NAC, respectively, on DEX-induced apoptosis in Ts65Dn thymocytes. At the end of the incubation period, cells were fixed with 1% paraformaldehyde, permeabilized with Triton X-100, and assessed for nuclear DNA content by PI staining in a flow cytometer. Data represent mean 6 SD of apoptotic cells in triplicate cultures for three independent experiments. ND, not done.

to block a defined molecule. Some inhibitors which were generally effective at low concentrations and nontoxic for intact cells were selected. DDC (at 5 mM), NAC (1–10 mM), vitamin E (only the 250 mM concentration was tested), N-propyl gallate (at 5 mM), and mannitol (5–20 mM) inhibited the DEX-induced apoptosis in Ts65Dn thymocytes (this effect was to some extent also observed in euploid thymocytes except for mannitol). Only marginal inhibition was observed with DMTU (10 –100 mM). Similarly, quercetin (up to 100 mM) and low concentrations of cysteamine (1 mM) did not protect. Similarly, antioxidants enzymes, catalase, or SOD (at 100 and 250 mg/ml, respectively) did not affect the percentages of apoptotic thymocytes. Finally, the addition of aminoguanidine (an inducible NO synthase inhibitor) had no effect, probably indicating that NO production was not involved in DEXinduced thymocyte apoptosis. To get an idea of the role of in vivo anti-oxidants on DEXdriven thymic PCD, the following experiment was performed. Six experimental groups of both euploid and Ts65Dn mice were treated or not (PBS injection) with antioxidants (NAC, DDC) for 2 wk. One day after i.p. injection of PBS or DEX, thymic apoptosis was evaluated in all animals. Results of a representative experiment are shown in Fig. 12. It seems that NAC and DDC were able to protect thymocytes from PCD and that this in vivo effect is equivalent to that observed in vitro.

Discussion Certain pathological conditions including infectious processes, tumor progression, inherited disorders, and other clinical entities are associated with thymus involution (30, 31, 39 – 41). Individuals with DS have also been reported to have abnormalities in their thymus (3, 24 –27). In a mouse model for DS, trisomy 16, the existence of hypoplastic thymus was reported. Unfortunately, trisomy 16 is incompatible with normal development, and only mouse fetuses could be studied, although the basis of the underlying mechanism was not clearly interpreted (4). Segmentally trisomic Ts65Dn mice fulfill the criteria for a DS model (5, 6), and

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FIGURE 12. Thymocytes are rescued in vivo from PCD with antioxidants. Different sets of euploid and Ts65Dn mice were injected i.p. with PBS or antioxidants (NAC or DDC, 100 mg/kg body weight, daily) for 2 wk. Then, they were injected i.p. with or without DEX (1 mg) as indicated. Thymuses were removed 24 h later and teased apart; cells were collected, processed as indicated in Materials and Methods, and assessed for nuclear DNA content by PI staining in a flow cytometer. Data represent the mean 6 SD of apoptotic cells in triplicate samples from a representative experiment of two performed.

this report represents our preliminary characterization of thymus dysfunction in these mice. PCD plays a crucial role in defining the T cell repertoire during T cell development in the thymus. Several studies in humans (42, 43) and mice (42, 44), concluded that deletion occurs at the CD41CD81 stage, that both CD41CD81 and CD42CD82 thymocytes die in situ via a PCD process, and that expression of Bcl-2 does not protect immature thymocytes from PCD. In Ts65Dn thymuses, the architecture is maintained. However, TUNEL staining demonstrated increased apoptosis in both cortex and medulla of such thymuses. Although apoptosis was always increased in Ts65Dn thymuses in comparison with age-matched normal controls, percentages of single- and double-positive and -negative subpopulations were maintained in constant ratios. Thymocyte subpopulations were screened for expression of Bcl-2 in our model, because some reports claimed that Bcl-2 could act as an endogenous antioxidant (45), and it seems that Bcl-2 antagonizes a relatively late step that could be defining an irreversible commitment point (46) However, others claimed that Bcl-2 function is an oxidative-independent process (47– 48). Within the apoptotic thymocytes, both control and Ts65Dn mice express similar percentages of the apoptotic regulatory protein Bcl-2. In addition, because .75– 80% of the apoptotic thymocytes are CD41CD81 and ,10% of this subpopulation express Bcl-2, our data could indicate that Bcl-2 is down-regulated at the CD41CD81 stage of development in both Ts65Dn and control thymocytes (42– 44, 49). The question that arises when extrapolating in vitro findings (DEX induces thymocyte apoptosis but LPS does not) to an in vivo situation (both agents induce apoptosis) is, “What could be the targets of LPS effects in the context of thymocyte apoptosis?” Available evidence suggests that the hypothalamic-pituitary-adrenal (HPA) axis, circulating cytokines, and activated macrophages are directly linked: 1) LPS injection enhanced in vivo production of various cytokines (i.e., IL-1, TNF-a, IL-6) via macrophage activation (J.-E. Paz-Miguel, R. Flores, L. Lo´pez de Rego, and F. Leyva-Cobiań, manuscript in preparation); 2) these cytokines stimulated the HPA axis, increasing circulating levels of adrenocorticotrophic hormone and corticosteroids (reviewed in Refs. 50 –52); 3) the rapid return of circulating cytokine concentrations to basal levels appears to be regulated by negative glucocorticoid feedback;

THYMOCYTE APOPTOSIS IN Ts65Dn MICE 4) on the other hand, it is well established that glucocorticoid hormones modulate T cell maturation in vivo.; 5) it seems that a precise balance of hormone concentration is crucial for T cell homeostasis in the thymus: low levels of hormones are required for appropriate T cell development, whereas higher glucocorticoid concentrations allow immature T cells to undergo increased apoptosis during systemic stress (53); and 6) it is considered that the glucocorticoids induce apoptosis in thymocytes by binding to a specific receptor belonging to the erbA oncogene-related steroid hormone receptor superfamily (54) and at least two glucocorticoidinducible genes (they encode two receptors that function as ATPand inositol 1,4,5-triphosphate-gated calcium channels) have been implicated in thymocyte apoptosis (reviewed in Ref. 55). Also, homeostatic control by the HPA axis is compromised with aging and in DS, leading to an increase in plasma adrenocorticotrophic hormone and corticosteroid levels (1, 52). This situation is theoretically operative in Ts65Dn mice. Probably under pathological situations (such as bacterial and viral infections), HPA axislinked thymus processes (e.g., PCD) could be exacerbated (as reported here by the experimental administration of a bacterial product, LPS, or a corticosteroid analogue, DEX). The absence of these complex influences on cultured thymocytes allows us to explain the virtual absence of LPS-induced apoptosis in vitro. During oxidative metabolism, harmful ROI are generated. These species are physiologically neutralized by antioxidant enzymes. First, SOD converts O22 to H2O2. Thereafter, catalase and glutathione peroxidase independently convert this to H2O (catalase, besides this activity, oxidizes formic acid and formate (56, 57)). However, the existence of increased SOD-1 activity in trisomic cells produce higher amounts of H2O2, and because this molecule is highly diffusible and convertible (via metal ion-driven HaberWeiss or Fenton reactions) to the most highly reactive zOH radical, oxidative damage and further lipid peroxidation of cell structures occur (28). Several ROI scavengers and inhibitors such as NAC, N-propyl gallate, and DDC significantly enhanced in vitro thymocyte survival from Ts65Dn mice and normal controls. NAC and DDC were also able to impair in vivo DEX-induced thymic apoptosis. Theoretically, the antioxidant effects of NAC could be exerted at two levels: 1) by direct reduction of ROI; and 2) by deacetylation, forming cysteine and consequently increasing glutathione production (12). This, in turn, acts as a substrate in the reduction of H2O2, catalyzed by glutathione peroxidase, to be converted to H2O. Finally, glutathione is an endogenous antioxidant that scavenges zOH radicals. However, DMTU, cysteamine, mannitol, and quercetin did not contribute to thymocyte rescue from PCD in Ts65Dn thymocytes as well as those from normal mice. DMTU reacts with H2O2 and HOCl (58) and although it could have contributed to the rescue of cells from PCD, the possibility that it may exert other biochemical effects cannot be ruled out. On the other hand, the observation that low concentrations of cysteamine do not protect against apoptosis was not surprising. It could indicate that processes where O22 participate are not dependent on triggering apoptosis. Mannitol is also an active scavenger of zOH radicals (12) but does not protect thymocytes. Similarly, quercetin, a competitive inhibitor of the ATP-binding site of phosphatidylinositol-3kinase, does not inhibit H2O2 production although it inhibits O22 formation (12). Finally, catalase and SOD had no effect, probably because they could not easily diffuse into the cells. The conclusion derived from studies with inhibitors is that results must be interpreted with caution, because the specificity of these scavengers is not absolute and varies according to cell type and apoptotic agent. For example: 1) staurosporine-induced apoptosis in cortical neurons from DS is not reversed by antioxidants (59); 2) NAC does

The Journal of Immunology not protect 7-M12 myeloid leukemic cells (which show a high intrinsic level of H2O2 production) from cycloheximide-induced apoptosis (60); 3) antioxidants show dual effects on oxidativelinked processing of proteins (12); and 4) catalase and SOD did not affect intracellular ROI in rat thymocytes, unlike NAC (61). Finally, it has been recently shown that ROI regulate signals involved in caspase activation and apoptosis (62). These authors elegantly demonstrated that T cells are rescued from O22 generation and cell death with a SOD mimetic, Mn(III)-tetrakis(5,10,15,20-benzoic acid)porphyrin (62). Our results support this observation. NO has also been associated with PCD of fresh thymocytes (63). Although the role of RNI in our model was not specifically addressed in this paper, it is probable that they do not contribute significantly to thymocyte apoptosis in Ts65Dn mice because of the following indirect observations: 1) NO22 concentration in peritoneal macrophages from Ts65Dn mice and controls was similar; and 2) aminoguanidine (an inducible NO synthase inhibitor) did not protect thymocytes from DEX-induced apoptosis. In addition, it has been reported that S-methylisothiourea (another inhibitor of NO production) had no effect on the PCD of activated T cells (62). Many studies have addressed the toxic role of zOH on mitochondria, cytoplasmic membranes, and lysosomes (38). It is probable that in our model, H2O2 could be participating in free radical formation, allowing the NADPH-dependent peroxidation to occur in subcellular structures through many possible interactions operating sequentially or simultaneously (37, 64 – 67). These mechanisms can induce protein and lipid peroxidation of key structural targets. 4-HNE and MDA are produced as major products of the peroxidative decomposition of v6-polyunsaturated fatty acids, the former being much more toxic than the latter (see Ref. 38 for a review). Generation of lipid peroxidation by-products clearly correlates with our observation that thymocyte apoptosis is induced by these compounds. Lipid peroxidation in thymocytes from Ts65Dn mice was greater than that in normal thymocytes, and DEX plays a collaborative role in vitro in the generation of harmful oxygen derivatives. As expected, DEX stimulates lipid peroxidation more than LPS. In consequence, lipid peroxidation could lead to increased membrane rigidity and other alterations in subcellular membranes that could contribute to apoptotic mechanisms. These observations agree with those indicating ROI generation also correlates with enhanced thymocyte apoptosis in the Ts65Dn model. Despite the fact that this issue is controversial, it is being generally accepted that mitochondria exert a decisive role in PCD, mainly for the following reasons: 1) inhibition of caspases does not always prevent irreversible apoptotic changes (62, 68); 2) PCD can be induced in cytoplasts (68); and 3) caspase inhibitors block DNA degradation but not PCD in activated T cells (62). Consequently, at least two pathways can operate simultaneously, separately, or sequentially to converge in irreversible T cell (and probably in cells from other lineages) death (62): 1) a direct caspase-dependent activation (e.g., through TNF-a or Fas signaling) and ROI-independent route; and 2) a ROI-dependent route together with a caspase-dependent activation needed for DNA degradation but not for PCD. Our data suggest that zOH plays a role in thymocyte apoptosis. These results also strongly indicate that ROI derived from H2O2 mediates the degeneration of thymocytes in Ts65Dn mice. These observations as well as those reporting severe early thymic involution in SOD-1 transgenic mice (28, 29) stress the relationship between apoptosis in both T cells and those of the thymic microenvironment and intracellular redox balance. However, their main molecular targets and downstream pathways are still undefined. In

5409 addition, Ts65Dn mice represent an interesting model with which to study regulatory pathways in the thymus in relation to: 1) the aging processes in general; 2) DS pathogenesis in particular; and 3) certain immunopathological processes (e.g., autoimmune disorders and leukemogenesis) associated with these conditions. Such studies are currently under way.

Acknowledgments We thank Jesu´s Florez and Ingrid M. Outschoorn for critical reading of the manuscript.

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