Changes in thymus size, cellularity and relation between thymocyte subpopulations in young adult rats induced by Somatostatin-14

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Neuropeptides Neuropeptides 41 (2007) 485–493 www.elsevier.com/locate/npep

Changes in thymus size, cellularity and relation between thymocyte subpopulations in young adult rats induced by Somatostatin-14 q - ergovic´, Ana K. Rakin, Ljiljana A. Dimitrijevic´, Danica M. Petrovic´-D Jasmina S. Ristovski, Natasˇa Z. Kusˇtrimovic´, Mileva V. Mic´ic´ * Immunology Research Center ‘‘Branislav Jankovic’’, Institute of Immunology and Virology ‘‘Torlak’’, Belgrade, Serbia Received 18 March 2007; accepted 29 June 2007 Available online 29 August 2007

Abstract The role of somatostatin on inhibition of both normal and tumor cell cycle, secretion of endocrine and exocrine cells, as well as induction apoptosis is well documented. However, its effect on T cell development and thymic structure is not fully clarified. In order to investigate the influence of somatostatin in vivo on the thymus structure and T cell development, the young adult Albino Oxford male rats were intracerebroventriculary treated with somatostatin-14. We examined the thymus compartments and its cellularity, through assessment of morphometric parameters by stereological method, and the relation between thymocytes subpopulations, over expression of CD4, CD8 and T-cell receptor (TCR) ab by flow cytometry. Additionally, we also determined the body and thymus weight of the rats, during the first three months of life, to define the time of SRIH-14 application. A decrease of relative thymus weight from the fourth weeks of postnatal life, and an unchanged relative thymus weight obtained in treated group indicates that SRIH-14 in young adult rats inhibits growth of whole organism, not only thymus. The changes in the absolute number and numerical density of cortical thymocytes indicate that SRIH-14 alters the true lymphoid tissue. SRIH-14 changes relation between thymocyte subsets, increase number of CD4CD8TCRab and CD4CD8+TCRabhi thymocyte subsets as well as the CD4CD8TCRablow/hi thymocytes, while decrease number of CD4+CD8+ TCRab/low/hi thymocyte subsets. These results indicate that somatostatin-14 is not involved in the control of the physiologic involution of the thymus, although induces thymic weight loss through the reduction of true lymphoid tissue. In addition, changes in frequency of thymocyte subpopulations, especially immature cells, indicate that SRIH-14 modulates thymocytes development and maturation.  2007 Elsevier Ltd. All rights reserved. Keywords: Rat; Thymus; T cells development; Somatostatin; Morphometry

1. Introduction The thymus is a primary lymphoid organ that provides a unique cellular and humoral microenvironment Abbreviations: DN, double negative; DP, double positive; SP, single positive; TCR, T cell receptor; SRIH, somatotropin release inhibiting hormone; TEC, Thymus epithelial cells; SSTR, Somatostatin receptor; PI, Propidium iodide; i.c.v., intracerebroventricularly. q The Serbian Ministry of Science and Technology grant supported this study, project No. 145049. * Corresponding author. Tel./fax: +381 11 467 465. E-mail address: [email protected] (M.V. Mic´ic´). 0143-4179/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2007.06.003

for the development of bone marrow-derived progenitors into immunocompetent T cells (Anderson and Jenkinson, 2001). The thymus microenvironmental cells (heterogeneous population of epithelial cells, macrophages and dendritic cells), are organized in the subcapsular, cortical and medullar regions (Jankovic´ et al., 1982; van Vielt et al., 1984; Bodey et al., 2000). These cells drive thymocytes development and maturation into immunocompetent T cells, through cell-to-cell contacts and via thymus hormones and other soluble factors (Jameson et al., 1995; Anderson and Jenkinson, 2001; Starr et al., 2003). Soluble factors that regulate T cell

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development are derived from at least two sources; thymic stromal cells that are the source of a variety of growth, differentiation, and survival factors, and neuroendocrone system as source of numerous factors that regulate thymopoiesis (Montecino-Rodriquez et al., 2005). In the physiological environment thymocytes are exposed to agents such as hormones and neuropeptides (Savino and Dardenne, 2000). One such is somatostatin (Somatotropin releasing inhibiting hormone, SRIH) a neuropeptide, widely distributed in the central nervous system, pituitary gland, gastrointestinal tract, pancreas, kidneys and lymphatic tissue (Reichlin, 1983). This neuropeptide exists in two biologically active isoforms, SRIH-14 and SRIH-28 (Krantic, 2000). It potently inhibits basal and stimulated secretion from many endocrine and exocrine cells, acts as a neurotransmitter/neuromodulator in the brain (Brazeau et al., 1972), and as a hormonal regulator of cell proliferation and differentiation (Pan et al., 1992; Muller et al., 1999). The biological effects of SRIH are initiated through its interaction with specific high-affinity transmembrane receptors, named sst15, expressed on the surface of responsive cells (Patel, 1999). The main physiological action of somatostatin is to inhibit release of GH (Corpas et al., 1993), anabolic hormone, involved in the thymic development and the various functions related to both the microenvironmental and lymphoid cells of this important organ (Savino and Dardenne, 2000; Sabharwal and Varma, 1996). On the other hand, presence of nerve endings, which can hold and release SRIH (Van Hagen et al., 1994), and SRIH producing cells, in medulla and cotico-medullary region of the thymus (Aguila et al., 1991; Leposavic´ et al., 1992), indicated on paracrine/autocrine route of SRIH activity in the thymus, through somatostatin receptors (SSTR) expression on thymocytes and thymic epithelial cells (Sedqi et al., 1996; Ferone et al., 1999, 2002; Solomou et al., 2002). The local production of SRIH in the thymus can be relevant since the neuropeptide could act at different levels, on different thymic cells and via different receptor subtypes (Ferone et al., 2000). Within the thymus, SRIH could be considered as a potent inhibitor of cell proliferation, over sst2 receptors (Bousquet et al., 2004), controller of secretory processes in the SRIH target cells (Nakahama et al., 1990), and inducer of cell apoptosis, via sst3 receptors (Sharma et al., 1996). It is previously described that SRIH-14, intracerebroventricularly (i.c.v.) applied, in adult male rats, decreased levels of GH, prolactine (PRL) and thyroid-stimulating hormone (TSH) (Milosevic et al., 1996), the anabolic hormones which play an integrating role in the growth, maintenance, repair, and functioning of the immune system (Savino and Dardenne, 2000). Moreover, we demonstrated previously that exogenous application of SRIH alters the thymus architecture and has an effect on the thymocyte subpopula-

tions, in peripubertal male rats (Petrovic-Djergovic et al., 2004). Considering that SRIH exerts different effects in the thymus, the present study was designed to evaluate whether repeated nanomolar doses of SRIH-14, applied immediately after puberty, change the thymus compartments and its cellularity, through assessment of morphometric parameters by stereological method. In addition, we examined the relation between thymocytes subpopulations, over expression of CD4, CD8 and T-cell receptor (TCR) ab, cell surface molecules, by flow cytometry. In order to define the time of SRIH-14 application, e.g. beginning the thymic atrophy, we determined the body and thymus weight of the rats, during the first three months of life.

2. Materials and methods 2.1. Rats Male AO strain rats, different of age (2-, 4-, 6-, 8-, 10-, 12- and 14-weeks-old) were used in the present study. The animals were kept under environmentally controlled conditions (12-h light/dark cycle, light on from 7 am until 7 pm; 22 C ± 1 C), and were provided with food and water ad libitum. Our Institutional Animal Care and Use Committee have approved the experimental protocol. 2.2. Animal preparation At the age of 10-weeks (young adult) the rats were subjected to operative procedure of cannulation, performed under total Nembutal anesthesia (35 mg/kg, intraperitoneally, Serva Feinbiochemica, Heidelberg), as we previously described (Petrovic-Djergovic et al., 2004). The silastic-sealed 20-gauge cannula (Starcˇevic´ et al., 1988) was inserted into a lateral brain ventricle, 2 mm laterally to the sagittal suture, 2 mm caudally to the frontal suture and 3.0 mm ventrally to the cortical surface. It was used for intracerebroventricular (i.c.v.) application of somatostatin. The cannula and a small stainless steel anchor screws were placed at a remote site on the skull and were fixed to the skull with dental acrylate (Simgal, Galenika, Belgrade). A minimal recovery time before beginning of the treatment was 5 days, as suggested (Myers, 1975). 2.2.1. Protocol After recovery, the rats were divided in two groups and each group was consisted of ten animals. The first group received i.c.v. three doses of 1-lg of SRIH-14 (Sigma Chemical, St Louis, USA, S-9129) dissolved in 5-ll saline, with 48-h-intervals between the treatments. The rats from the second group received 5-ll saline in

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the same manner as experimental rats. Animals were sacrificed by decapitation 24 h after last injection. The examination of position and permeance of the cannula was accomplished on the day of the sacrifice by vital dyes. Only animals with confirmed position in the lateral ventricle were considered for analysis.

section of the gland, from each rat, at a magnification of 125/1000·, using the multipurpose test system M 42 (Weibel, 1979). For each investigated structure, the size of the final sample was determined, within the confidence interval of 95%, using the equation:

2.2.2. Preparation of thymus cell suspensions The thymuses were carefully removed, weighted and used for a single-cell suspensions preparation, by grinding the thymus tissues between the frosted ends of microscope slides, in cold phosphate-buffered saline (PBS, pH 7.3) containing 2% heat-inactivated fetal calf serum (FCS, Gibco, Grand Island, NY, USA) and 0.01% sodium azide (NaN3, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). The obtained singlecell suspensions were passed through the fine nylon mesh, washed three times in cold buffer solution, counted by a standard haemocytometer, and adjusted the cell concentration to 1 · 107 cells per ml. The cells viability, as determined by Trypan blue exclusion, was routinely greater than 95%.

(n-number of test areas necessary for analyses, y-variation of attained values from confidence limits (±5%), s-standard deviation, x-arithmetically mean sample). The absolute number of the cortical/medullar thymocytes was calculated from the numerical density of thymocytes in the outer and deeper cortex/medulla and the volume of the cortex/medulla. Absolute volume of the thymus was calculated on the basis of their weight assuming an average specific gravity of 1.039 (Swinyard, 1938).

2.3. Flow cytometry Triple immunofluorescence labeling was performed for detecting thymocyte CD4, CD8 and TCRab expression. The thymocyte suspensions (1 ± 0.5 · 106/100 ll) were incubated simultaneously with fluorescein-isothiocyanate (FITC)-conjugated anti-CD4 (W3/25, Serotec, UK), phycoerythrin (PE)-conjugated anti-CD8 (MRC OX-8, Serotec) and biotin-conjugated anti-TCRab (R73, Serotec) monoclonal antibodies, 30 min at 4 C in the dark. After three washings in PBS solution, cells were incubated with streptavidin-peridinchlorophyll protein (Streptavidin-PerCP) (BD Bioscience, USA), under the same conditions. In the next step, after another washing in PBS solution, single-cell suspensions were fixed in paraformaldehyde and kept in the dark at 4 C until analysis by FACScan flow cytometer. Usually 5 · 104 cells per sample were analyzed by FACScan Research Software program (Becton Dickinson). Nonspecific IgG isotype-matched controls were used for each fluorochrome type to define background staining. Dead cells and debris were gated out on the basis of forward and side scatter. 2.4. Morphometry Extracted thymuses were snap frozen in cryostat embedding media (Reichert, Wien, Austria). The frozen sections (5-lm thickness), stained with hematoxylin and eosin were analyzed by Olympus BH2 microscope. The volume of the thymic cortex/medulla and numerical density of thymocytes in the volume unit (lm3) of the thymus compartments were determined on every tenth

n ¼ ð200=y  s=xÞ2 ðKalisnik; 1985Þ

2.5. Statistical analysis The data obtained from each rat were averaged per group and standard deviation of the mean values was calculated. Mean values were compared by nonparametric Mann–Whitney test using SPSS 10 for Windows software package. The results were expressed as the mean value ± SD, and differences at p < 0.05 were accepted as the level of significance.

3. Results 3.1. Thymus weight and thymocytes yield The results showed visibly an increase of body and thymus weight, during the first three months of life. Namely, the absolute thymus weight increased very fast from the second week of postnatal life until puberty (approximately eight weeks), and subsequently the thymus weight began to descend (Fig. 1a). The relative thymus weight, the thymus weight with respect to body weight, was visibly diminished from the fourth weeks of postnatal life (Fig. 1b). In young adult animals SRIH-14 conspicuously diminished of the body weight and the absolute thymus weight (p < 0.05), while the relative thymus weight was unchanged (Fig. 2). The total yield of thymocytes in SRIH-14 treated group were significantly lower (p < 0.05) than in corresponding control animals (Fig. 2). 3.2. Analysis of CD4/CD8 expression on the thymocytes Analysis of relative proportion of thymocyte subpopulations, defined by expression of CD4 and CD8 surface markers, revealed that SRIH-14 alters the relation

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Fig. 1. The thymus and body weights (a), and relative thymus weights (i.e. thymus weight with respect to body weight) (b) of male AO rats. The results are presented as mean ± SD. n = 7–10.

among thymocyte subpopulations. Such it increased (p < 0.01) the percentages of double negative (DN) cells and both single positive (SP) thymocyte subpopulations, while reduced the relative proportion of double positive (DP) thymocytes (p < 0.05; Fig. 3a and b), compared with adequate control. When number of thymocytes within thymocyte subpopulations were related to thymus weight, we found that SRIH-14 increases the absolute number of DN cells (p < 0.05), and the number thymocytes in both of SP subpopulations, slightly, while reduces (p < 0.01) absolute number of cells within DP subpopulation (over 100%) (Fig. 3c). 3.2.1. Analysis of TCRab expression on the thymocytes In the present study, we estimated the level of TCRab expression, using R73 mAbs against constant determinant of the rat ab heterodimeric TCR (Hu¨nig et al., 1989). Expression of TCRab, was evaluated as high level of TCRab expression (TCRabhi), low, but noticable level of TCRab expression (TCRablow), and undetectable level TCRab expression (TCRab) (Tsuchida et al., 1994). In respect to the expression of CD4 and CD8 molecules, and the level of TCRab expression, twelve subsets of thymocytes were described and the number of these thymocytes subsets was estimated. In the experimental group of rats, the number of the cells expressing TCRabhi was unchanged, but the number of TCRab/low expressing cells was significantly decreased (p < 0.001 for TCRab cells; p < 0.01 for TCRablow cells; Fig. 4; Table 1). The relative proportion

Fig. 2. Effects of SRIH-14 on the body and thymus weight, relative thymus weight and absolute number of thymocytes. Results are representative of 2-independent experiments. Values represent mean ± SD; *p < 0.05; vs. control.

of these subsets was in control 3.7: 3.4: 1.3, in treated rats 1.9: 2.3: 1.2, respectively. In SRIH-14 treated rats, the number of thymocytes with undetectable level of TCRab within DN was significantly increased (p < 0.05), number of DPTCRab cells was significantly decreased (p < 0.001), while the number of both SPTCRab thymocyte subpopulations was unchanged (Fig. 4, Table 1). SRIH-14 significantly rises number of DNTCRablow (p < 0.001) thymocytes, reduces (p < 0.01) of DPTCRablow cells number, while the number of cells within both SPTCRablow thymocyte subpopulations was unchanged (Fig. 4, Table 1). The number of DP thymocytes expressing TCRab at high density (TCRabhi) was lower (p < 0.01) than in corresponding control. The number of DNTCRabhi cells (p < 0.05) and SPCD8+TCRabhi thymocytes (p < 0.05) was increased, but increase of SPCD4+TCRabhi cells was unsignificant (Fig. 4, Table 1). 3.3. Morphometry Analysis of thymus sections by light microscopy demonstrated that SRIH-14 induces constriction of thy-

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Fig. 3. Two-color analysis of CD4 and CD8 expression on thymocytes from control and SRIH-14 treated rats. Expression of CD4 was detected by direct immunofluorescence with FITC, and expression of CD8 was detected by direct immunofluorescence with PE. Ordinate represent fluorescence intensity of PE, abscissa, fluorescence intensity of FITC. The percentages of CD4  CD8 + (up left), CD4 + CD8+ (up right), CD4  CD8-(down left) and CD4 + CD8-(down right) cells are represented on corresponding corner. Table (b) represents the number of cells within thymocytes subsets. Results are representative of 2-independent experiments. Values represent mean ± SD; *p < 0.05; **p < 0.01; vs. control.

mus cortex with scant cell number (Fig. 5). Considering the importance of thymus compartments in the process of the thymocytes differentiation, we investigated whether application of SRIH-14 evoked morphometrical changes in the thymus compartments. In young adult rats, SRIH-14 reduces of the thymic cortex volume (0.27 ± 0.01 cm3; vs. 0.35 ± 0.02 cm3 in controls; p < 0.05) and absolute number of thymocytes within the thymic cortex (7.15 · 108 ± 1.05 · 108; vs. 4.52 · 108 ± 0.27 · 108; p < 0.01). However, the thymus medulla volume and number of thymocytes in the thymus medulla, were not altered (Fig. 6). It is evident, from above results, that the cortex volume and number of thymocytes within the cortex were included in the reduction of thymus size in the SRIH-14 treated rats. In order to explain which part of cortex participated in alteration of cortical thymocytes number, we determined numerical density of thymocytes in outer and deeper thymic cortex. The results demonstrated that SRIH14 significantly decreased the numerical density of thymocytes within both parts of the thymic cortex. The decrease the numerical density of thymocytes was on level of signification p < 0.05 in the outer, apropos, p < 0.01 in the deeper cortex (Fig. 6).

4. Discussion In this study, intracerebroventricular route of SRIH14 application was selected because this way enables

avoidance of rapid peptide decomposition in the systemic circulation, provides unobstructed approach of the peptide to the cerebral structures and bypassing the blood-brain barrier (Gmerek et al., 1983). Previous reports indicate that thymic atrophy starts with puberty (Dominguez-Grepe and Rey-Mendeze, 2003). Moreover, it has been proposed that two groups of hormones, one causing thymic hypertrophy (GH) and insulin-like growth factor-I (IGF-I), and the other producing thymic atrophy (sex hormones) (Aspinall and Andrew, 2001) can be associated with thymic atrophy. Principal support of the hypothesis that hormones contributes to thymic atrophy is based on studies demonstrating an increase of thymic cellularity after GH and IGF-I administration, in old mice and mice with congenitally hormonal deficiency (Murphy et al., 1992; Bar-Dayan and Small, 1994; Montecino-Rodriguez et al., 1998), and gonactomized rats (Leposavic´ et al., 2002). If the hypothesis that changes in the production of above mentioned hormones trigger or sustain thymic involution correct, than an assumption is that SRIH14, as potent inhibitor of secretion in majority of the pituitary cell, applied immediately after puberty, should contribute thymus atrophy. However, an unchanged relative thymus weight (relation thymic and body weight), obtained in this study indicate that SRIH-14 in young adult rats inhibits growth of whole organism, not only thymus. Our data are in line with the results of Min et al. (2006) that suggest that age-related changes in

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Fig. 4. TCRab expression on thymocytes within the four major thymocyte subpopulations from control and SRIH-14 treated rats. Thymocytes were stained in three-color and first analyzed on the basis of CD4 and CD8 expression, than were further examined for the expression of TCRab. The relative proportion of TCRab expression was assessed at three distinct levels: negative, low and high. Vertical scale cannot be compared because they were modified to allow visualisation of small populations. The histograms, obtained by analysing 2 · 104 cells, are representative of 2-independent experiments.

the endocrine system do not underline thymic involution. In addition, a decreases of relative thymus weight from the fourth weeks of postnatal life (Fig. 1b), suggests that SRIH applied immediately after puberty, probably is not included in thymus involution. Discrepancy of these results with our previous data, decrease of relative thymus mass, obtained in peripubertal rats (Petrovic-Djergovic et al., 2004), can be in relationship with ages of rats. Our results, also suggest that SRIH-14, although is not included in the thymus atrophy, most likely is involved in thymic weight loss. A decrease of thymic cellularity, thymic cortex volume and thymocytes numerical density obtained in this study, support this notion. The change in the absolute number and numerical density of cortical thymocytes indicates that SRIH-14 alters the true lymphoid tissue. Having in mind, neuropeptides involvement in the regulation of the thymus stromal component function (Savino and Dardenne, 2000), it is clear that further investigations of stromal cortex component is necessary for determination whether stromal cells of the cortical region contribute to cortex volume reduction. On the other hand, the absence of significant changes in the volume and cellularity of thymus medulla, as numerical density of thymocytes, after SRIH-14 treatments, can be related with rise of SPTCRabhi thymocytes number. Taking to the consideration the subsets of cells defined by expression of TCRab, we observed that SRIH-14 decreases the number of TCRab/low cells, but unchanges population of TCRabhi thymocytes. These findings suggest that SRIH have effect on the immature thymocytes subsets. A significantly decrease of DPTCRab thymocyte, slightly decrease of SPTCR ab cells, precursors of DP cells (Matsumoto et al., 1991), and increase of DNTCRab subset indicates that SRIH-14 alters of DN to DP transition. Inhibition of proliferation of normal and tumor cell by somatostatin via sst2A receptor (Pan et al., 2004; Bousquet et al., 2004) expressed on immature thymocytes, proliferating cells (Ferone et al., 2002; Petrovic´-Djergovic´ et al., 2007), and loss of cells with unsuccessfully rearrangement of b chain, can be involved in this alteration. In addition, decrease of DPTCRablow thymocytes, cells that have to go throughout the positive selection process (Huang et al., 1996), suggests that a big part of the most immature cells either undergo apoptosis or do not use conventional process of positive and negative selection. The decrease of DPTCRabhi thymocyte subset, post-positive selection cells, intermediate between DPTCRablow and SPTCRabhi thymocytes (Jameson et al., 1995), and increasing of single positive cells, suggests sensibility of thymocytes in hereby stages of maturation on SRIH-14 as well as involvement of this neuropeptide in the acceleration of the positive selection. Similar results about cellularity, changes in thymic

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Table 1 Absolute number of cells within thymocyte subsets of control and SRIH-14 treated rats defined by CD4, CD8 and TCRab expression Subpopulations

Number/thymus · 106 Control rats

SRIH-14-treated rats

Total TCRab Total TCRablow Total TCRabhi

367.35 ± 12.5 343.09 ± 20.9 126.36 ± 5.9

191.07 ± 11.51*** 232.10 ± 8.2** 125.40 ± 7.7

CD4-8-TCRab CD4-8-TCRablow CD4-8-TCRabhi

17.57 ± 4.2 1.34 ± 0.33 2.76 ± 0.07

22.49 ± 7.1* 8.96 ± 0.2*** 3.63 ± 0.2*

CD4 + 8 + TCRab CD4 + 8 + TCRablow CD4 + 8 + TCRabhi

333.05 ± 3.34 335.47 ± 2.51 28.78 ± 3.35

154.82 ± 11.5*** 218.73 ± 19.8** 14.41 ± 3.3**

CD4  8 + TCRab CD4  8 + TCRablow CD4  8 + TCRabhi

14.23 ± 1.5 2.85 ± 0.4 32.3 ± 1.34

11.99 ± 0.85 2.9 ± 0.5 42.84 ± 4.3*

CD4 + 8  TCRab CD4 + 8  TCRablow CD4 + 8  TCRabhi

2.34 ± 0.03 4.10 ± 0.8 62.17 ± 9.04

1.87 ± 0.5 2.97 ± 0.02 64.51 ± 1.5



Results represent the mean ± S.D. from 2-independent experiments. * p < 0.05. ** p < 0.01. *** p < 0.001.

compartments and relations between thymocyte subpopulations have been obtained in experiments with SRIH-28 applied in the same manner (Petrovic´-Djergovic´ et al., 2007). However, analyses of thymocytes subsets defined by TCR expression revealed that SRIH-14 modulates postselectional maturation into single positive thymocytes towards SPCD8+TCRabhi, while SRIH-28 has an opposing effect on mentioned subset. Different distribution and different effects of two somatostatin biologically active forms in the same tissues/organs like pancreas, gastrointestinal tract etc. has been described in other tissues/organs (Amherdt et al., 1987; Francis et al., 1990; Patel, 1999; Srikant and Patel, 1981) and arises the question does this two somatostatin biological form act on the same or different thymic cell types. It should be, also, emphasized that SRIH-14 increase a minor subset of DN cells expressing TCRab+ (TCRa blow/hi), pathway of development is still poorly defined. These cells represent a distinct lineage cells, except the main ab lineage pathway, in adult mice, which does not pass conventional processes of positive and negative selection (Budd and Mixter, 1995). DNTCRab+ thymocytes arise from DP cells with high-avidity TCR signal, bordering on negative selection, which escape negative selection and down regulate of CD4 and CD8 co-receptor molecules (Budd and Mixter, 1995). They are not detectable until 3 week of adult life. The physiological function of DNTCRab+ cells is not yet revealed for sure. In co-culture with naive T cells and antigen, these cells express immunoregulatory activ-

Fig. 5. Photomicrographs thymus tissue sections stained by hematoxylin and eosin from control (a) and experimental (b) young adult male rats, demonstrate contracted thymus cortex induced by SRIH-14 (b; Bar = 70 lm) and decreased cellularity of the thymus cortex.

ity (Wang et al., 2002). Presence of DN T regulatory cells in the spleens and lymph nodes of thymectomized mice, irradiated and reconstituted with T cell-depleted bone marrow cells, suggest that functional DN T regulatory cells may preferentially develop outside of the thymus (Ford et al., 2006). These authors suggest that DN T regulatory cells may represent a developmentally and functionally unique cell population, as well as that this cells are not derived from CD8+ T cell precursors. However, by model described in normal and ‘‘autoreactive’’ TCR-transgenic mice, DNTCRab+ cells bear a demethylated CD8a gene, previously express CD8 and pass positive selection by MHC class I or class-I like molecules binding to CD8. The previous expression of CD8 may be functionally important to subsequent development of cells DN phenotype (Takahama et al., 1991). Within the DNTCRab+ fraction of thymocytes T cells bearing self-reactive TCR Vb determinants (Takahama et al., 1991) and subset of thymocytes undergoing apoptosis (Kersh and Hedrick, 1995) are detected.

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College of Medicine, Hershey, PA, USA for providing us with somatostatin.

References

Fig. 6. Stereological changes in the thymuses of young adult male rats treated with SRIH-14. Changes of stereological parameters are shown as graphics: volumes of the thymus compartments (a), total thymocyte numbers (b) and numerical density of thymocytes (c) in the various thymus compartments. Results are representative of 2-independent experiments. Values represent mean ± S.D. **p < 0.01; *p < 0.05; vs. control.

Finally, these results indicate that SRIH-14 is not the critical factor involved in initiation the thymus atrophy after puberty, although decreases thymic weight as consequence reduction of true lymphoid tissue. Its modulatory effect on the T cell development, especially immature cells, may be of great value for its therapeutic use.

Acknowledgements The Serbian Ministry of Science and Technology grant support this study, project No. 145049. The authors are grateful to W.B. Severs, Ph.D., Department of Pharmacology, Pennsylvania State University,

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