Age-associated changes in CD90 expression on thymocytes and in TCR-dependent stages of thymocyte maturation in male rats

Age-associated changes in CD90 expression on thymocytes and in

TCR-dependent stages of thymocyte maturation in male rats

Gordana Leposavic a,b,*, Vesna Pesic b, Dusko Kosec a, Katarina Radojevic a,

Nevena Arsenovic-Ranin c, Ivan Pilipovic a, Milica Perisic a, Bosiljka Plecas-Solarovic b

a Institute of Immunology and Virology ‘Torlak’, Immunology Research Center ‘Branislav Jankovic’, Belgrade, Serbia and Montenegrob Department of Physiology, Faculty of Pharmacy, Belgrade, Serbia and Montenegro

c Department of Immunology and Microbiology, Faculty of Pharmacy, Belgrade, Serbia and Montenegro

Received 18 October 2005; received in revised form 1 March 2006; accepted 7 March 2006

Available online 2 May 2006

Abstract

To elucidate the effects of ageing on T-cell-maturation, in 3- and 18-month-old rats, we analysed the expression of: (i) CD4/CD8/TCRab and

(ii) Thy-1, which is supposed to be a regulator of TCRab signalling, and thereby the thymocyte selection thresholds. Since an essential role for

TCRab signalling in the development of CD4C25CTreg-cells was suggested, the frequency of these cells was also quantified. We demonstrated

that, as for mice, early thymocyte differentiational steps within the CD4-8- double negative (DN) developmental stage are age-sensitive.

Furthermore, we revealed that TCRab-dependent stages of T-cell development are affected by ageing, most likely due to an impaired expression

of Thy-1 on TCRablow thymocytes entering selection processes. The diminished frequency of the post-selection CD4C8C double positive (DP)

cells in aged rats, together with an overrepresentation of mature single positive (SP) cells, most probably suggests more efficient differentiational

transition from the DP TCRabhigh to the SP TCRabhigh developmental stage, which is followed by an increase in pre-migration proliferation of the

mature SP cells. Moreover, the study indicated impaired intrathymic generation of CD4C25CTreg-cells in aged rats, thus providing a possible

explanation for the increased frequency of autoimmune diseases in ageing.

q 2006 Elsevier Inc. All rights reserved.

Keywords: Ageing; T-cell differentiation; CD90 expression; Thymocyte apoptosis; ConA; CD4C25C thymocytes

1. Introduction

Empirical data on a substantial increase of morbidity and

mortality due to infectious diseases in old age (Grubeck-

Loebenstein, 1997; Aspinall, 2000) suggest a decline in the

efficiency of immune system function with age. In the same

line are findings of diminished protective immunity following

prophylactic vaccination against influenza, and re-emergence

of such latent infections as Varicella zoster in aged individuals

(Globerson and Effros, 2000). Moreover, an increase in the

frequency of cancer and autoimmune diseases was also found

to accompany ageing (Wick et al., 2000). However, contrary

to the classical view of ‘immunosenescence’ as the age-

associated decline in immune response, there is an emerging

0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2006.03.006

* Corresponding author. Address: Department of Physiology, Faculty of

Pharmacy, 450 Vojvode Stepe, 11151 Belgrade (KumodraZ), Serbia and

Montenegro. Tel.: C381 11 39 70 379; fax: C381 11 46 74 65.

E-mail address: [email protected] (G. Leposavic).

consensus that immunological ageing is more related to a

complex reorganization than to a simple decline in immune

system functions (Globerson and Effros, 2000; Romanyukha

et al., 2003).

The involution of the thymic lymphoepithelial component is

one of the most prominent features of ageing in the immune

system. Based on data available in animals and humans, it is

generally accepted that the volume of true thymic tissue

(excluding the perivascular space, and the adipose and fibrous

tissue) attains maximum size at puberty, after, which it

gradually decreases (Fabris et al., 1988; George and Ritter,

1996; Bodey et al., 1997; Shanker, 2004). Therefore, with

ageing, the thymic tissue weakens as a source of naive T

lymphocytes (Brill et al., 1990; Consolini et al., 2000;

Romanyukha et al., 2003). The reduced T-cell output, together

with an increase in apoptosis of naive T-cells limits the ability

of aged individuals to respond to newly encountered antigens.

The markedly reduced size of the naive T-cell subpopulation,

together with an increased number of memory cells in the

periphery, is a clear-cut characteristic of ageing in the immune

system (Romanyukha et al., 2003).

Experimental Gerontology 41 (2006) 574–589

www.elsevier.com/locate/expgero

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589 575

It has been supposed that the decline in thymic function with

ageing is a consequence of changes at both the prethymic

thymocyte precursor level and the intrathymic stromal cell

level (Hirokawa et al., 1986; Yu et al., 1997; Mackall et al.,

1998). The underlying mechanisms of age-related thymic

involution have not been fully depicted yet, although clear

experimental support for both non-genetic, i.e. hormonal

(Kelley et al., 1986; Fabris et al., 1988, 1997; Li et al., 1992;

Hirokawa et al., 1997; Leposavic et al., 2002), neural (Madden

et al., 1997) and genetic mechanisms (Lau and Spain, 2000;

Hsu et al., 2005) exists. Moreover, in accordance with the well-

documented symbiotic relation between the developing T

lymphocytes and the thymic nonlymphoid component (Ritter

and Boyd, 1993), it has been suggested that the age-related

alterations in thymocyte development contribute to thymic

involution (Lau and Spain, 2000).

Multifaceted age-related changes in both the multiple-step

process of intrathymic T-cell maturation and its intrathymic

regulation have been assumed. The earliest thymocytes

lack functional TCR, as well as CD4 and CD8 (Michie and

Zuniga-Pflucker, 2002; Zamoyska and Lovatt, 2004). Within

this stage thymocytes express an invariant pre-Ta protein and

rearrange the TCRb locus. Successful rearrangement of the

TCRb locus precedes its covalent pairing with the pre-Tachain. This is known as b selection, while subsequent

association with CD3 produces the functional pre-TCRacomplex (Michie and Zuniga-Pflucker, 2002; Zamoyska and

Lovatt, 2004). These cells are signalled to proliferate and turn

on expression of the CD4 and CD8 genes, becoming double

positive (DP) cells (Jameson et al., 1995; Guidos, 1996; Michie

and Zuniga-Pflucker, 2002; Zamoyska and Lovatt, 2004).

Thymocytes unable to rearrange their TCRb locus successfully

are deleted by apoptosis. Following TCRa locus rearrange-

ment, the thymocytes interact with epithelial cells expressing

an optimised set of self-peptides within the MHC complex.

From this point, the thymocyte will survive if signals generated

by TCR engagement by MHC/peptide are interpreted as

appropriate (positive selection), but will be deleted by

apoptosis if the generated signals are interpreted as either

inappropriately weak (death by neglect) or inappropriately

strong (negative selection) (Jameson et al., 1995; Guidos,

1996; Michie and Zuniga-Pflucker, 2002; Zamoyska and

Lovatt, 2004). The positively selected thymocytes upregulate

TCRab expression and downregulate one of the coreceptors,

either CD4 or CD8, to give the mature MHC-restricted self-

tolerant CD4C or CD8C single positive (SP) mature T-cells

(Jameson et al., 1995; Guidos, 1996; Zamoyska and Lovatt,

2004). Since, thymocytes progress through phenotypically

well-defined steps during maturation, T-cell maturation can be

monitored by analysis of the distribution of thymocyte subsets.

Some age-sensitive maturational steps during early thymocyte

maturation have been clearly identified in mice (Brill et al.,

1990; Yehuda et al., 1998). Moreover, it has been suggested

that later, TCR-dependent, checkpoints in T-cell development

may be also altered in the course of ageing in mice (Lau and

Spain, 2000). However, data related to the age-associated

changes in T-cell maturation in rat thymus are rather limited.

Therefore, this study was designed in order to identify subtle

thymocyte differentiational steps, which are affected by ageing,

with special focus on the late TCRab-dependent steps. To

reach this goal (i) the surface density of TCRab on thymocytes

in respect to CD4/CD8 expression and (ii) expression of Thy-1,

which is supposed to be a regulator of TCRab signalling, and

thereby of the thymocyte selection thresholds (Hueber et al.,

1997; Killeen, 1997), were examined. The rationale for the

analysis of Thy-1 is provided by data indicating that the age-

associated decline in thymus weight and thymic cellularity

leads to an increase in the density of noradrenergic nerve fibres

in the parenchyma and noradrenaline concentration in the

thymus (Madden et al., 1997), on the one hand, and by the

observation that exogenous cAMP and noradrenaline can

induce decreases in steady state Thy-1 mRNA levels in

T-lineage cells through posttranscriptional destabilization of

Thy-1 mRNA (Wajeman-Chao et al., 1998), on the other hand.

In addition, thymocyte apoptosis and proliferation, events

associated with thymocyte maturation, were also analysed.

Having in mind: (i) the age-related increase in the frequency

of self-reactive lymphocytes and autoimmune diseases (Wick

et al., 2000; Shanker, 2004) and (ii) that under normal

circumstances, some self-reactive T-cells fail to be eliminated

in the thymus and escape into the periphery where they are

exposed to continuous control by a dominant T-cell-mediated

protective mechanism produced by intrathymically generated

regulatory T-cells (Treg-cells) that express CD4 and CD25

(Seddon and Mason, 2000; Shevach, 2002; Godfrey et al.,

2000), one may assume that in aged individuals the

autoreactive T/Treg-cell balance is affected due either to an

absolute or a relative decline in the thymic production of Treg-

cells. To explore this hypothesis the relative number of CD4C25C cells in the thymus of both young adult and aged rats was

quantified.

2. Materials and methods

2.1. Animals

Male Wistar rats were purchased from the Military Medical

Academy Animal House, Belgrade, housed in polyethylene

cages containing sterilized wood shavings and given rodent

chow and tap water ad libitum. They were housed in a room

maintained at a temperature of 21G2 8C, with a 12 h light/dark

cycle. Rats were euthanized under ether anaesthesia by

exsanguination. The experimental protocol was approved by

the Institutional Animal Care and Use Committee.

Rats were euthanized at the age of either 3 (6 rats) or 18

months (5 rats). Animals that showed overt signs of illness,

including low body weight, tumours or splenomegaly were

excluded from the study. Each thymus was aseptically isolated,

trimmed of all excess body fat and gently blotted on sterile

gauze to remove excess blood. The two lobi were divided and

individually weighed. The left lobus was fixed for stereological

analysis, while the right lobus was dissociated by gently

grinding the tissue on a sterile 60-mm sieve screen in complete

RPMI-1640 medium. The single-cell suspension thus obtained

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589576

was washed three times in ice-cold complete RPMI-1640

medium. The cells were then counted in an improved Neubauer

hemocytometer and the number of cells per milligram thymic

tissue, and consequently the total number of cells per thymus,

were estimated. The viability of such cell preparations,

determined by Trypan blue exclusion, was routinely greater

than 95%.

2.2. Chemicals, antibodies and immunoconjugates

Sodium azide, Merocyanine (MC540) and Concanavalin A

(Con A) were purchased from Sigma–Aldrich Chemie GmbH,

Taufkirchen, Germany. Dexamethasone (Dexason, Dex) was

obtained from Galenika A.D., Zemun, Serbia and Montenegro.

RPMI 1640 powdered medium (Sigma–Aldrich Chemie

GmbH) was dissolved in redistilled water according to the

manufacturer’s instructions. To prepare complete RPMI

medium, 2 mM L-glutamine (Serva, Heidelberg, Germany),

1 mM sodium pyruvate (Serva), 100 units/ml penicillin (ICN,

Costa Mesa, CA, USA), 100 mg/ml streptomycin (ICN) and

10% fetal calf serum (FCS) (Gibco, Grand Island, NY, USA)

were added. FCS was inactivated by heating the serum at 56 8C

for 30 min.

For immunolabeling the following monoclonal antibodies

(mAbs): phycoerythrin (PE)-conjugated anti-CD4 (clone

W3/25, Serotec, Oxford, UK), fluorescein-isothiocyanate

(FITC)-conjugated anti-CD8 (clone MRC OX-8, Serotec),

peridinin chlorophyll protein (PerCP)-conjugated anti-TCRab(clone R73, BD Biosciences Pharmingen, Mountain View, CA,

USA), biotin-conjugated anti-CD25 (clone MRC OX-39,

Serotec), FITC-conjugated anti-CD44 (Pgp-1) (clone OX-49,

Serotec), FITC-conjugated anti-CD161 (clone 10/78, Serotec),

FITC-conjugated anti-CD90 (Thy-1.1) (clone HIS 51, BD

Biosciences Pharmingen), anti-CD3 (clone G4.18, BD

Biosciences Pharmingen) and-RT6.1 (clone P4/16, Serotec)

were used as the first-step reagents. As the second-step

reagents, streptavidin-PerCP, streptavidin-PE, FITC-conju-

gated F(ab 0)2 rabbit anti-rat IgG purchased from Serotec,

were applied, respectively. The appropriate IgG isotype

controls were obtained from BD Biosciences Pharmingen.

2.3. Tissue processing and stereological analysis

After fixation in Bouin’s solution and dehydration in a

graded series of ethyl alcohol, the thymic specimens were

embedded in paraffin, serially cut into 5 mm thick sections,

which were then stained with haematoxylin and eosin. Every

40th section (approximately 20 sections per organ) was

subjected to analysis of the volume of lymphoepithelial thymic

tissue, denoted as true thymic tissue (George and Ritter, 1996;

Shanker, 2004), and thymic cellularity.

Stereological measurements were performed by a point

counting method (Weibel, 1979) using image analysis software

(Micro Image, Version 4.0, OLYMPUS). The test areas were

randomly chosen, and each image, acquired using a digital

camera, was saved, overlaid with the corresponding grid and

analysed.

Absolute volumes of the cortical and medullary lymphoe-

pithelial thymic compartments, and interlobular, subcapsular

and perivascular connective and adipose tissues, were

estimated from the volume of the processed and embedded

organ and the volume density (Vv) of the corresponding

compartment (the percentage of whole organ volume occupied

by the analysed structure). During processing and embedding

the thymic tissue shrunk by approximately 34% in both groups,

and this was determined stereologically, as previously

described (Plecas-Solarovic et al., 2004). Vv of each thymic

compartment was determined under !40 magnification, using

an orthogonal test grid with 130 points, according to the

equation: VvZPf/Pt (Pf represents the number of test points

hitting the analysed structure, while Pt is the total number of

test points falling on the organ). The total number of analysed

test-areas was 100 per animal.

The total number of thymocytes/thymic lobus was

calculated from the numerical density (Nv) of thymocytes, as

the number of cells per unit volume, and the absolute volume of

the lobus (Plecas-Solarovic et al., 2004). From the total number

of thymocytes/lobus the number of thymocytes/mg thymic

tissue, and consecutively the total number of thymocytes/

thymus, were calculated. The Nv of thymocytes was estimated

under immersion magnification, using a grid corresponding to

the multipurpose M42 test-system (Weibel, 1979). For each

thymic compartment 60 test areas per animal were measured.

Nv of thymocytes was determined according to Weibel and

Gomez (Weibel, 1979).

2.4. Phenotyping of lymphocytes by flow cytometry

analysis (FCA)

Immunolabeling of the cell suspensions was performed as

previously described (Leposavic et al., 2005). Briefly, aliquots

of 1!106 lymphoid cells in 100 ml RPMI 1640 medium were

centrifuged at 350g for 5 min, at 4 8C, to yield a pellet. The

cells were incubated for 30 min on ice in the dark with

fluorochrome-conjugated mAbs (direct labelling) or with

fluorochrome-conjugated and biotin-conjugated mAbs

(indirect labelling), and then washed three times in the

medium. When biotin-conjugated mAbs were applied, the

cells were incubated again for 30 min on ice with appropriate

fluorochrome-conjugated streptavidin. After labelling the cells

were washed twice in the medium, and then in ice-cold

phosphate buffered saline (PBS, pH 7.4) containing 0.01%

sodium azide. The cells were then fixed in 0.5 ml of 1%

paraformaldehyde in PBS and kept at 4 8C in the dark until

analysis. Twenty-thousand cells per sample were analysed

using a FACScan flow cytometer (Becton Dickinson, Mountain

View, CA, USA). Non-specific IgG isotype-matched controls

were used for each fluorochrome type to define background

staining, while dead cells and debris were excluded from

analysis by selective gating based on anterior and right-angle

scatter. The percentage of positive cells for each labelling was

determined using the CellQuest software (Becton Dickinson).

In addition, CD90 expression was analysed by estimation of the

mean channel number, which represents density of the surface

ged rats

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589 577

marker expression. The mean channel number was determined

for the thymocytes from both young adult and aged rats, and

the relative change in the mean intensity of fluorescence (MIF)

was calculated as follows:

MIF for histogram of young adult ratsKMIF for histogram of a

MIF for histogram of aged rats

(Kamath et al., 1998).

2.5. Detection of apoptotic thymocytes

Since, thymocytes are normally rapidly eliminated by

phagocytes in vivo, the relative number of apoptotic cells

was quantified after 18 h of cultivation, as previously suggested

(Kamath et al., 1998). One hundred microliters of freshly

isolated thymocytes at a concentration of 5!106 cells/ml were

plated in 96-well flat-bottom plates (Nunc A/S, Roskilde,

Denmark) in RPMI 1640 complete media. To these cells,

100 ml of complete medium containing Dex was added to attain

a final concentration of 1 mM Dex, a dose known to induce

apoptosis of rat thymocytes in vitro (Brown et al., 1993). In

control cultures 100 ml of complete medium was added. Cells

were harvested after 18 h of culture for analysis of the relative

numbers of apoptotic cells. After the estimation of cell viability

by Trypan blue dye exclusion, MC 540 and FITC-conjugated

Annexin V/propidium iodide (PI) assays were performed.

2.5.1. Detection of apoptotic thymocytes using lypophilic

dye MC 540

The staining was performed according to the procedure

described by Mower et al. (1994). For labelling, just before

FACS analysis, 5 ml of 1 mg/ml stock MC540 solution in

double-distilled H2O was added to 1 ml of thymocyte

suspension containing 106–107 cells. The stock solution of

MC540 was filtered through a 0.22 nm filter and stored in

the dark at 4 8C no longer than 1 month. All samples were

analysed on a FACScan flow cytometer by CellQuest

Software (Becton Dickinson). According to the intensity of

MC540 fluorescence, on the one hand, and forward scatter,

on the other hand, (Cohen, 1991), three subsets of

apoptotic cells can be distinguished: (1) cells in early

apoptosis, (2) cells in advanced apoptosis and (3) cells in

late apoptosis/necrosis. It has been reported that apoptotic

cells remain MC540 bright until their DNA content reaches

a certain point and then display decreasing levels of

MC540 staining (Reid et al., 1996). Thus, generally, the

cells in late apoptosis/necrosis are characterized by low

Table 1

Thymus weight, thymus weight/body weight ratio, volume of true thymic tissue, num

3- and 18-month-old Wistar rats

Absolute thymus

weight (mg)

Thymus weight/

body weight (%)

Volume o

thymic tis

3-Month-old 570.2G31.1 1.67G0.07 336.6G1

18-Month-old 482.5G104.2 0.7G0.1** 123.6G3

The data are expressed as meanGSEM (nZ5–7). *p!0.05, **p!0.01.

forward scatter and an undetectable level of MC540

binding.

2.5.2. Detection of apoptotic thymocytes using Annexin

V/propidium iodide

Thymocyte apoptosis was also examined using an Annexin

V/PI apoptosis detection kit (BD Biosciences Pharmingen).

Apoptotic cell staining was performed according to the

producer’s manual. Twenty thousand cells per sample were

analysed using a FACScan flow cytometer (Becton Dickinson)

and CellQuest software (Becton Dickinson). Since, it has been

shown that: (i) normal thymocytes undergoing spontaneous

apoptosis in vitro initially appear as Annexin VC/PI- cells and

(ii) the same cells, with increasing culture duration, become

Annexin VC/PIC cells (Okasha et al., 2001), Annexin VC/

PI- single positive cells are considered early apoptotic, whereas

Annexin VC/PIC double positive cells are believed to be in a

late stage of apoptosis rather than in necrosis (Okasha et al.,

2001;Vermes et al., 1995). Annexin VK/PIC single positive

cells are reckoned to be mainly dead cells (Okasha et al., 2001;

Vermes et al., 1995).

2.6. Detection of BrdUC cells

Bromodeoxyuridine (BrdU) incorporation was utilized to

identify DNA-synthesizing cells in vitro. A total of 2!105

cells/well (100 ml) of thymocytes from young adult or aged rats

were dispersed into plastic 96-well plates (Nunc A/S) and

cultured for 48 h at 37 8C in a 5% CO2 humified air atmosphere

without or with 2.5 mg/ml ConA in a total volume of 200 ml

complete RPMI 1640 culture medium. All cultures were run in

triplicate. During the last 18 h of culture the cells were pulsed

by 1 mM BrdU/well. For the detection of BrdU incorporation

into cells, the BrdU/7-AAD Flow kit (BD Biosciences

Pharmingen) was used. 7-AAD labelling enabled detection of

the late apoptotic/necrotic hypodiploid cells and subsequently

their exclusion from the analysis. To determine the phenotypic

characteristics of the BrdUC cells, the staining with FITC-

conjugated anti-BrdU Abs and 7-AAD was combined with

immunolabelling with biotin-conjugated anti-CD3 mAbs as the

first step-reagent, and streptavidin-PE as the second-step

reagent. The staining was performed according to the

BrdU/7-AAD flow kit producer’s manual. All samples were

ber ot thymocytes/mg thymic tissue and total number of thymocytes/thymus in

f true

sue (mm3)

Thymocyte number

(!106)/mg thymic tissue

Total thymocyte

number/thymus (!108)

9.2 1.47G0.25 8.41G0.53

9.4* 0.24G0.06** 1.21G0.45*

Fig. 1. In panel A are shown representative dot-plots of MC540 labeled thymocyte cultures from (left dot-plots) 3- and (right dot-plots) 18-month-old rats in presence

(CDex) and absence of dexametasone (KDex). According to the intensity of MC540 thymocyte staining and forward scatter (FSC), three subsets of cells at

distinct phases of apoptosis (early apoptosis, advanced apoptosis and late apoptosis/necrosis) are delineated. The relative proportions of all apoptotic cells

and cells in different phases of apoptosis in CDex and KDex thymocyte cultures from 3- and 18-month-old rats are shown in (B) upper and (C) lower histogram,

respectively. The data are expressed as meanGSEM (nZ5–7). *p!0.05, **p!0.01. All the SEM values less than 0.1 are omitted. a3 months of age -Dex vs. 3

months of age CDex; b18 months of age -Dex vs. 18 months of age CDex; c3 months of age -Dex vs. 18 months of age -Dex; d3 months of age CDex vs. 18 months

of age CDex.

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589578

Fig. 2. In panel A are shown representative dot-plots of double Annexin V/PI stained thymocytes from (a, b) 3- and (c, d) 18-month-old rats cultured (a, c)

without (KDex) or (b, d) with dexamethasone (CDex). The total number of apoptotic cellsZcells in the lower right quadrant (cells in early apoptosis)Ccells

in the upper right quadrant (cells in advanced apoptosis)Ccells in the upper left quadrant (cells in late apoptosis/necrosis). In the histograms are shown the

relative proportions of (B) all apoptotic cells and (C) cells in different phases of apoptosis in KDex and CDex thymocyte cultures from 3- and 18-month-old

rats. The data are expressed as meanGSEM (nZ5–7). **p!0.01. All the SEM values less than 0.1 are omitted. a3 months of age KDex vs. 3 months of age CDex; b18 months of age KDex vs. 18 months of age CDex; c3 months of age KDex vs. 18 months of age KDex; d3 months of age CDex vs. 18 months of

age CDex.

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589 579

Fig. 3. Relative proportions of BrdUC cells in the thymocyte cultures from 3-

and 18-month-old rats, as determined by flow cytometric analysis of BrdU

incorporation. The thymocytes were cultured for 48h in medium without ConA

(KConA) or with ConA (CConA). The data are expressed as meanGSEM

(nZ5–7). **p!0.01. All the SEM values less than 0.03 are omitted. a3 months

of age KConA vs. 3 months of age CConA; b18 months of age KConA vs. 18

months of age CConA; c3 months of age KConA vs. 18 months of age

KConA; d3 months of age CConA vs. 18 months of age CConA.

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589580

analysed on the FACScan flow cytometer by CellQuest

software (Becton Dickinson).

2.7. Statistical analysis

Data are expressed as mean (X)Gstandard error of the mean

(SEM). To assess the significance of differences between

means, the Mann–Whitney U-test was applied using the

program, SPSS 10.0 for Windows. PR0.05 was considered

significant.

3. Results

3.1. Age-associated changes in thymus weight and total

number of thymocytes

There was no significant difference in the absolute weight of

the thymuses from 3- and 18-month-old rats. However, the

relative thymic weight (ratio to body weight) was significantly

(p!0.01) diminished in aged rats and its average value was

2.4-fold less than that in young adults (Table 1). Furthermore,

stereological analysis revealed that the average volume of

processed true thymic tissue was 2.7-fold (p!0.05) lower in

aged compared with young adult animals (Table 1). This

decrease in the volume of true thymic tissue reflected, at least

partly, a marked reduction in thymus cellularity. The number

of thymocytes per milligram of thymic tissue, and conse-

quently the total number of thymocytes per thymus, were

significantly reduced in aged rats compared with young adults

as determined by both direct counting of the cells in the

thymocyte suspension (0.16G0.06!106/mg thymic tissue in

aged vs. 1.81G0.23!106/mg thymic tissue in young adult rats,

p!0.05 and 0.79G0.17!108/thymus in aged vs. 10.74G1.26!108/thymus in young adult rats, p! 0.01) and

sterological analysis (Table 1).

3.2. Influence of ageing on thymocyte apoptosis

The reduced number of thymocytes may reflect: (i)

decreased ingress of T-cell precursors into the thymus; (ii)

increased elimination of thymocytes by apoptosis and (iii)

decreased proliferation of developing T-cells. To assess the

putative contribution of increased thymocyte apoptotic

elimination to the observed reduction in thymus cellularity,

not only spontaneous thymocyte apoptosis, but also the

sensitivity of thymocytes to apoptotic signalling induced by

glucocorticoids, as well known inducers of thymocyte

apoptosis, was examined. Using both MC540 and Annexin

V/PI assays it was demonstrated that the frequency of apoptotic

cells, in the absence of known inducers of apoptosis, was

significantly (p!0.01) greater in the cultures of thymocytes

from aged than in those from young adult rats (Figs. 1 and 2).

Furthermore, it was demonstrated that this increase in apoptotic

cells from aged rats reflected a drastic (p!0.01) rise in the

relative frequency of cells in late apoptosis/necrosis, while the

relative numbers of cells in earlier phases of apoptosis did not

significantly differ between cultures of thymocytes from rats of

different age (Figs. 1 and 2). This finding may indicate that

thymocytes from aged rats in culture underwent spontaneous

apoptosis earlier than those from young adult rats.

As we expected, both the assays for detection of apoptotic

cells demonstrated that Dex in the cultures of thymocytes

caused a significant (p!0.01) rise in the relative number of

apoptotic cells irrespective of the age of animals from, which

they were isolated (Figs. 1 and 2). However, to our surprise, in

the presence of Dex, the frequency of apoptotic cells in the

cultures of thymocytes from aged rats was significantly (p!0.01) lower than that in cultures of thymocytes from young

adults. Furthermore, the Dex-induced increase in the percen-

tage of apoptotic cells in cultures of thymocytes from young

adult rats reflected a significantly (p!0.01) increased

frequency of cells in early and advanced apoptosis, while

that in aged rats was associated with a significantly (p!0.05

and 0.01 by MC540 and Annexin V/PI staining, respectively)

increased frequency of cells in late apoptosis/necrosis (Figs. 1

and 2). Although, the overall number of apoptotic cells in

DexC cultures of thymocytes from aged rats was significantly

(p!0.01) lower than that in cultures of thymocytes from young

adult rats, it seems that these cells underwent apoptosis earlier

in the presence of Dex.

3.3. Influence of ageing on BrdU incorporation in thymocytes

In order to clarify, the mechanisms underlying the reduced

cellularity of thymus from aged animals, we attempted not only

to identify cells within thymocyte subpopulations delineated by

the surface density of TCR, which were actually involved in

DNA replication, but also to assess the proliferative potential

of these cell subpopulations. For this purpose BrdU incorpor-

ation in DNA was tested in cultures of thymocytes from young

adult and aged rats in the absence and presence of ConA. In the

absence of ConA the frequency of BrdUC was significantly

(p!0.01) lower in the thymocyte cultures from aged rats than

in those from young adults (Fig. 3). ConA evoked significant

increases in the frequency of BrdUC cells in the thymocyte

cultures from rats of both ages. However, ConA was

more effective in increasing the relative number of cells

incorporating BrdUC in cultures of thymocytes from young

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589 581

rats (the average number of BrdUC cells increased by 38%)

than in those from aged rats (the average number of BrdUCcells increased by 28%).

Further analysis of the BrdU incorporation into cells within

distinct thymocyte subsets delineated by quantitative differ-

ences in CD3 surface expression, showed that, irrespective of

ConA presence, the percentage of BrdUC cells within all

subsets of CD3 cells (CD3K, CD3low and CD3high) was

significantly (p!0.01) reduced in aged rats. In the thymocyte

cultures from young adult rats ConA significantly (p!0.05)

increased the frequency of BrdUC cells within both subsets of

cells expressing detectable levels of CD3 (Fig. 4). However, in

the thymocyte cultures from aged rats ConA caused a

significant (p!0.01) increase in the relative number of

BrdUC cells only within the CD3high thymocyte subset,

and this increase was more pronounced (the average number

of BrdUC cells increased by 52%) than in young adult rats

(the average number of BrdUC cells increased by 22%)

(Fig. 4).

3.4. Influence of ageing on distribution of thymocyte subsets

Having in mind that, along the intrathymic maturational

route from thymocyte precursors to mature T-cells, subsets at

distinct stages of maturation may be distinguished by a

characteristic constellation of CD4/8/TCRab differentiational

markers, it is clear that changes in intrathymic T-cell

development may be monitored by assessing the distribution

of the thymocyte subsets. Therefore, to elucidate putative age-

related changes in intrathymic T-cell maturation, the relative

sizes of thymocyte subsets delineated by the expression of

coreceptor molecules and the surface density of TCRab were

quantified.

3.4.1. CD4/8 expression on thymocytes

The analysis of CD4/8 expression on thymocytes demon-

strated that the relative number of CD4C8C DP cells

was significantly (p!0.01) diminished in aged rats, while

Fig. 4. Relative proportions of BrdUC cells within different cell subsets gated accord

cultures from 3- and 18-month-old rats. The thymocytes were cultured for 48 h in me

as meanGSEM (nZ5–7). *p!0.05, **p!0.01. All the SEM values less than 0.2 we

age -ConA vs. 18 months of age CConA; c3 months of age -ConA vs. 18 months

CD4-8- DN cells (p!0.01) and SP (CD4C8- and CD4-8C)

cells were significantly (p!0.01 and 0.05, respectively)

increased when compared with young adult rats (Figs. 5 and 6).

3.4.2. TCRab expression on thymocytes

The expression of TCRab was evaluated using R73 mAbs

that are, most likely, directed at a constant determinant of the rat

ab heterodimeric TCR (Hunig et al., 1989). Since, thymocytes

at different stages of differentiation have already been shown to

express distinct surface levels of the CD3/TCRab complex

(Shortman et al., 1991; Tsuchida et al., 1994), the thymocytes

with undetectable TCRab (hence referred to as TCRabK),

TCRablow and TCRabhigh expression were gated, as indicated

in Fig. 5. Compared with young adult rats, in 18-month-old rats a

significant (p!0.01) decrease in the frequency of TCRabK

thymocytes followed by a proportional increase (p!0.01) in

that of TCRabhigh cells was found. The frequency of TCRablow

cells remained unaltered in aged rats (Figs. 5 and 6).

As previously observed (Tsuchida et al., 1994; Leposavic

et al., 2005), by gating cells according to the quantity of surface

TCRab and plotting CD4 and CD8 expression against each

other, four subsets may be delineated by CD4/8 expression

within each gate (Fig. 5).

3.4.2.1. TCRabK. The analysis of phenotypic characteristics of

TCRabK cells, with respect to CD4 and CD8 expression,

revealed that the relative number of the least mature CD4-8-

DN cells (p!0.01) and CD4-8C SP thymocytes (p!0.05)

were significantly increased while that of CD4C8C DP cells

was significantly (p!0.01) diminished (Figs. 5 and 6).

3.4.2.2. TCRablow thymocytes. Although, the relative number

of TCRablow thymocytes (Figs. 5 and 6) was not

significantly altered in aged rats, significant changes in the

distribution of TCRablow thymocyte subsets were detected.

In rats of both ages, the majority of cells within this gate

displayed the CD4C8C DP phenotype. Cells of this

phenotype have been shown to enter the selection process

ing to the intensity of CD3 staining (CD3K,CD3low, CD3high) in the thymocyte

dium without ConA (KConA) or with ConA (CConA). The data are expressed

re omitted. a3 months of age -ConA vs. 3 months of age CConA; b18 months of

of age -ConA; d3 months of age CConA vs. 18 months of age CConA.

Fig. 5. Three-colour flow cytometric analysis of thymocytes from (Panel A) 3- and (Panel B) 18-month-old rats stained with anti-CD4PE, anti-CD8FITC and anti-

TCRab PerCP mAbs. In the upper rows of the each panel flow cytometric profiles of CD4/CD8 (middle dot-plot) and TCRab (right histogram) staining of cells

within R1 region (left dot-plot) are shown, while low-er rows of the each panel represent CD4/CD8 expression on TCRabK,TCRablow, TCRabhigh thymocytes. The

gates for TCRabK, TCRablow and TCRabhigh cells were set as it is shown in upper right histograms.

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589582

(Shortman et al., 1991; Jameson et al., 1995; Guidos, 1996;

Michie and Zuniga-Pflucker, 2002; Zamoyska and Lovatt,

2004) and their relative number was significantly (p!0.01)

reduced in aged rats compared with young adults (Figs. 5

and 6). However, the relative numbers of cells belonging to

the other TCRablow thymocyte subsets were significantly

(p!0.01) increased in aged rats (Figs. 5 and 6).

3.4.2.3. TCRabhigh thymocytes. Compared with young adults,

in 18-month-old rats the relative number of post-selection

Fig. 6. Relative proportions of thymocytes delineated by expression of CD4/CD8 (A) and TCRab (left histograms within B, C and D panels), as well as relative

numbers of TCRabK (Panel B, right histogram), TCRablow (Panel C, right histogram), TCRabhigh (Panel D, right histogram) thymocyte subsets delineated by

CD4/CD8 expression in 3- and 18-month-old rats. The data are expressed as meanGSEM (nZ5–7). *p!0.05, **p!0.01. All the SEM values less than 0.4 were

omitted.

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589 583

CD4C8C DP cells representing the intermediate stage of

development between the CD4C8C DP TCRablow and

the most mature SP TCRabhigh (Jameson et al., 1995;

Guidos, 1996; Michie and Zuniga-Pflucker, 2002;

Zamoyska and Lovatt, 2004) was slightly, but significantly

(p!0.01) decreased. However, the frequencies of their

descendents, the most mature medullary CD4C8- and

CD4-8C SP cells were significantly (p!0.01) increased in

the thymus of aged rats (Figs. 5 and 6). The frequency of

cells belonging to the small subset of CD4-8- DN

thymocytes did not differ between the two groups of rats

(Figs. 5 and 6).

3.5. Thymocyte expression of CD161

In an attempt to explain the increase in frequency of CD4-8-

DN TCRab- cells in aged rats the expression of the NK cell

marker, CD161, on thymocytes was analysed. The results

showed that the frequency of CD161C cells was slightly, but

significantly (p!0.05) increased in aged (3.16G0.07%)

compared with young adult rats (2.74G0.08%). Thus, this

increase contributed to the rise in frequency of CD4-8- DN

TCRab- cells in aged rats. However, as the ab T-cell

lineage consists of distinct subsets, one of which is NKT

characterized by expressing both NK and T lymphocyte

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589584

markers (Godfrey et al., 2000; Pear et al., 2004), to answer

whether an increase in the relative number of NKT (CD161CTCRabC) cells contributed to the rise in CD161C cells in

aged rats, the relative number of these cells was quantified, as

well. However, there was no significant difference in the

relative proportion of NKT-cells between aged (2.08G0.29%)

and young adult (1.60G0.07%) rats.

3.6. Influence of ageing on thymocyte expression of CD90

Since, we demonstrated a reduction in relative numbers of

CD4C8C DP TCRablow cells entering the TCR-dependent

selection processes (Chan et al., 1993; Jameson et al., 1995;

Cantrell, 2002) and selected CD4C8C DP TCRabhigh cells

(Jameson et al., 1995; Guidos, 1996) in aged rats, and as CD90

(Thy-1) is reckoned to be a signalling molecule involved in the

regulation of TCRab signalling (Killeen, 1997; Hueber et al.,

1997), the expression of CD90 on thymocytes from both aged

and young adult rats was analysed.

The relative number of thymocytes with an undetectable

level of CD90 (Thy-1) was dramatically (p!0.05) increased in

aged rats (20.37G2.44%) compared with that in young adult

rats (1.85G0.21%). In addition, MIF for CD90 was markedly

decreased in aged rats, as its value in young adult rats was

Fig. 7. (A) Representative overlaid histogram of the surface expression of CD90 b

matched control is shown by dotted line (MIF for CD90 staining on thymocytes from

rats is given in the right corner of the histogram). (B) MIF for CD90 staining on thy

relative proportions of thymocytes with low (CD90low), intermediate (CD90int) and h

The data are expressed as meanGSEM (nZ5–7). *p! 0.05; **p!0.01. All the S

396G12% greater than in aged rats. MIF indicates the density

of antigen expression on a per cell basis, and it has been shown

that a 100% greater MIF represents a two-fold greater the

density of surface antigen (Kamath et al., 1998). Accordingly,

the average density of CD90 expression was almost fivefold

greater in young adult than in aged rats. Taking into account

the surface density of CD90, three subsets of CD90C cells

(CD90low, CD90int and CD90high) were delineated (Fig. 7). In

aged rats, the relative number of CD90low cells was

significantly (p!0.01) increased, while those of CD90int and

CD90high cells were significantly (p!0.01) reduced.

In an attempt to elucidate the mechanisms underlying the

reduction in frequency of post-selecton CD4C8C DP

TCRabhigh cells (Shortman et al., 1991; Jameson et al., 1995;

Guidos, 1996), the surface expression of CD90 by TCRablow

thymocytes was examined. The relative number of TCRablow

thymocytes with undetectable CD90 levels did not significantly

differ between aged (1.96G0.65%) and young adult (1.25G0.08%) rats. However, in young adult rats MIF for CD90 was

markedly greater (383.2G13.3%) than in aged rats suggesting

that the average surface density of CD90 per thymocyte of

TCRablow phenotype was almost fivefold greater (Fig. 8).

Furthermore, within the TCRablow subset of thymocytes from

aged rats CD90high cells were not detected (Fig. 8). In the same

y thymocytes from 3- (broken line) and 18-month-old rats (solid line). Isotype

18-month-old rats vs. MIF for CD90 staining on thymocytes from 3-month-old

mocytes from 3- and 18-month-old rats. (C) Percentages of CD90C cells, and

igh (CD90high) surface density of CD90 expression in 3- and 18-month-old rats.

EM values less than 0.14 were omitted.

Fig. 8. Flow cytometric analysis of the CD90 surface expression by TCRablow thymocytes from (C) 3- and (D) 18-month-old rats (MIF for CD90 staining is given in

the right corner of each histogram). The gates for TCRablow cells were set as it is shown in histograms (A) and (B), respectively. (E) MIF for CD90 staining on

TCRab low thymocytes from rats of both ages. (F) Relative proportions of TCRablow cells expressing CD90 at low (CD90low), intermediate (CD90int) and high

density (CD90high) in 3- and 18-month-old rats. The data are expressed as meanGSEM (nZ5–7). **p!0.01. All the SEM values less than 0.09 were omitted. n.d.,

not detected.

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589 585

population of cells a significant (p!0.01) drop in the relative

number of CD90int cells followed by an increase (p!0.01) in

the frequency of CD90low cells was found (Fig. 8).

3.7. Influence of ageing on the relative number

CD4CCD25C thymocytes

Because, it has been shown that: (i) the thymus, besides

production of conventional T-cells, generates CD4C Treg cells

that are capable of preventing the development of experimen-

tally induced autoimmune diseases (Shevach, 2002), and (ii)

CD25 is a useful marker for the detection of these cells in the

thymus, the relative number of CD4C25C cells was examined

in rats of both ages. It was found that the frequency of these

cells was significantly (p!0.05) diminished in aged rats

compared with young adults (Fig. 9). Furthermore, since

activated mature T-cells possess the capacity to migrate to the

thymus (Agus et al., 1991), we attempted to answer whether the

decreased relative number of CD4C25C cells in aged rats

reflected a reduced size of the thymocyte subpopulation with a

regulatory phenotype. To this end, having in mind that RT6

expression is restricted to the final stages of postthymic T-cell

development, so that this antigen is not expressed on thymic

lymphocytes and postthymic antigenically naive T-cells

(Mojcik et al., 1991), we estimated the frequency of CD4C25CRT6- thymocytes in rats of both ages. It was shown that

the frequency of CD4C25CRT6- cells was also significantly

(p!0.01) reduced in aged rats compared with young adults.

Fig. 9. Flow cytometric analysis of CD4/CD25 expression by (panel A) all and (panel B) RT6K thymocytes from (left dot-plots) 3- and (right dot-plots) 18-month-

old rats. Upper single parameter histograms in panel B represent RT6 expression in (left) 3- and in (right) 18-month-old rats. Single-parameter overlaid histograms

represent CD4 expression by (Panel A) all and (Panel B) CD25CRT6K thymocytes from 3- (white histogram) and 18-month-old rats (gray histogram). Relative

proportions of the all CD4CCD25C and CD4CCD25CRT6K thymocytes in rats of both ages are shown in right histograms in Panel A and Panel B, respectively.

The data are expressed as meanGSEM (nZ5–7). *p!0.05; **p!0.01.

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589586

This finding speaks in favour of a reduction in the relative

contribution of the cells expressing regulatory CD4C25Cphenotype in aged rats.

4. Discussion

This study has demonstrated a sharp drop in the cellularity

and total volume of lymphoepithelial thymic tissue in 18-

month-old Wistar rats compared with their 3-month-old

counterparts. However, in spite of this the thymic weight did

not significantly differ between these two groups of rats due to

an age-related increase in the volume of nonlymphoid, i.e.

adipose and connective tissue. Contrary to this finding, an age-

related reduction in the overall thymus weight was reported in

many strains of rats and mice (Fabris et al., 1988; Li et al.,

1992; Bodey et al., 1997; Leposavic et al., 2002). This

discrepancy may be related to a strong genetic contribution

influencing age-related thymic changes, for example, Buffalo

strain rats do not experience thymic involution (Hirokawa

et al., 1994). It has been suggested that: (i) reduction in the

volume of thymic lymphoepithelial tissue produces a radical

decrease in the number of recent thymic emigrants and (ii) the

functional capacity of the residual true thymic tissue correlates

with its anatomical measurements (Pawelec et al., 1999). The

drop in thymus cellularity with age is fully consistent with the

findings in the cultures of thymocytes from aged rats showing:

(i) the accelerated apoptotic elimination of thymocytes and (ii)

the reduced frequency of cells incorporating BrdU spon-

taneously or in a response to ConA stimulation. The reduced

frequency of apoptotic cells in response to Dex in thymocyte

cultures from aged rats compared with that in thymocyte

cultures from young adults may be explained by a significant

reduction in the relative numbers of DP cells, which are

believed to be the most sensitive to glucocorticoid-induced

apoptosis (Nieto et al., 1992). The present data on apoptotic

and proliferative characteristics of thymocytes from aged rats

in culture are in accordance with those showing that

thymocytes from aged mice and rats exhibit an increase in

the expression of cell death genes, i.e. p53, bax and caspase 3

(Kapasi and Singhal, 1999) and a significant decrease in

spontaneous and mitogen-induced proliferation (Kaplan and

Garvey, 1981; Yu et al., 1997; Globerson and Effros, 2000; Hsu

et al., 2002, 2005; Tian et al., 2003), respectively.

The study has also shown substantial changes in the

distribution of thymocyte subsets delineated by expression of

CD4/8/TCRab in aged rats compared with young adults

suggesting age-associated alterations in thymocyte develop-

ment. In accordance with previous work (Capri et al., 2000) an

increased frequency of the least mature CD4-8- DN TCRabK

cells was found in aged rats. The relative number of these cells

has been also reported to increase in the thymus of aged mice

(Aspinall, 1997; Thoman, 1997; Yehuda et al., 1998; Hsu et al.,

2002). The present findings, similarly to those for mice

(Ales-Martinez et al., 1988), indicate that the increase in the

frequency of CD4-8- DN TCRabK cells in aged rats partly

reflected an overrepresentaion of NK cells in the thymus of

aged animals compared with young adults. Therefore, a

possible contribution of some other mechanisms such as:

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589 587

(i) an increased entry of precursor cells; (ii) reduced apoptotic

loss of these cells and (iii) their increased proliferation, to the

increase in frequency of CD4-8- DN TCRabK cells in aged

rats should be also taken into consideration. There are no data

to support the first option. On the contrary, it appears that there

is no significant age-dependent loss in the number of bone-

marrow-resident pre-T precursor cells (Sharp et al., 1990), and

it has been suggested that ‘seeding’ of the aged thymic

reticulum by blood–borne precursors is similar to that of the

young thymus (Donskoy and Goldschneider, 1992; Thoman,

1995, 1997; Foss et al., 2001; Shanker, 2004). There are also no

data to support the contribution of either reduced apoptotic loss

or increased cell proliferation to the age-associated increase in

the frequency of CD4-8- DN TCRabK cells. The increase in

frequency of these cells in the thymus of aged mice has been

ascribed to developmental blocks that occur with differentia-

tive transition within the CD4-8- DN TCRabK developmental

stage itself and to decreased progression from this to the next

CD4C8C DP stage (Aspinall, 1997; Thoman, 1997; Yehuda

et al., 1998; Capri et al., 2000; Hsu et al., 2002). The hereby

reported reduction in the frequency of BrdUC cells within

CD3K subsets of thymocytes from aged rats in culture seems to

be consistent with the previous assumption indicating that the

transition of thymocytes through differentiation steps within

the DN stage becomes limited leading to a restriction in the

number of cells capable of proliferation and further transition

to the DP stage. In keeping with this assumption is the

reduction in the relative numbers of CD4-8- DN TCRabK

descendent CD4C8C DP TCRabK cells in aged rats.

However, a reduced frequency of CD4C8C DP TCRablow

cells in aged rats, in the light of a decreased relative number of

their immediate CD4C8C DP TCRabK precursors, on the

one hand, and a reduced relative number of their CD4C8C DP

TCRabhigh descendants, on the other hand, may suggest,

not only reduced thymocyte transition from the DN to DP

differentiational stage, but also less efficient positive selection

and/or overexaggerated negative selection. The dramatically

reduced surface density of Thy-1 on TCRablow thymocytes

(encompassing mainly CD4C8C DP cells) from aged rats

found here favours this assumption, since it has been

demonstrated that thymocytes from ThyK/K mice exhibit

inappropriate negative selection due to a lack of Thy-1-

mediated negative regulation of TCRab signalling and

activation thresholds during thymocyte differentiation (Hueber

et al., 1997). Several other findings in the present study also

favour this hypothesis. Firstly, the decreased frequency of

BrdUC cells within the CD3low subset in thymocyte cultures

from aged rats in the presence of ConA, which is shown to

induce positive selection and thereby survival and further

maturation of DP thymocytes (Lovatt et al., 2000; Pongracz

et al., 2003), seems to be consistent with the previous

assumption. Secondly, the increased frequency of apoptotic

cells in thymocyte cultures from aged rats compared with those

from young adult rats is also in keeping with the same

hypothesis. Finally, it has been reported that the relative

number of Thy 1.1C thymocytes is diminished in 24-month-

old female Wistar–Furth rats but could be augmented after

implantation of GH3 pituitary adenoma cells (Kelley et al.,

1986).

The increased frequency of SP (CD4-8C and CD4C8-)

TCRabhigh thymocytes in aged rats, together with the reduced

frequency of their CD4C8C DP TCRabhigh predecessors,

may indicate more efficient differentiation of the positively

selected cells. In favour of this option are data indicating that

when there is a developmental block in T-cell development,

reparatory mechanisms in the thymus increase the efficiency of

generation of mature SP cells facilitating events beyond the

block (Almeida et al., 2001). Furthermore, it may also suggest

an increase in pre-migrational proliferation of these cells and/

or increased re-entry of T-cells from the periphery. Having in

mind that, differently from immature T-cells, ConA causes

activation and proliferation of mature T-cells, the first option is

supported by data showing that ConA evoked a more

pronounced increase in the frequency of BrdUC cells within

the mature CD3high subpopulation in thymocyte cultures from

aged rats than in those from young adults. The increased

frequency of cells showing the TCRabCCD90- mature

peripheral phenotype (Williams, 1989), in the thymuses from

aged rats (data not shown) is fully consistent with the second

option. Furthermore, it should be pointed out that the increased

frequency of mature SP TCRabhigh cells in the thymus may

reflect decreased export of these cells into the periphery.

However, other findings suggesting that the ‘efficiency’ of

thymus cell export increases with low numbers of thymus cells,

but insufficiently to compensate for the reduced production of

mature thymus cells, speak against the latter option (Almeida

et al., 2001).

Finally, providing that, in aged rats, there is an increased

frequency of CD4C re-entrants in the thymus as has been

suggested for mice (Mehr et al., 1996), collectively taken, the

present findings on alterations in thymocyte subsets in aged rats

(increased frequencies of the least mature CD4-8-TCR- cells

and the most mature CD4C8-TCRabhigh cells, but decreased

frequency of DP cells) seem to fit the framework of a

previously stated hypothesis that T-cell maturation is subject to

intrathymic feedback regulation. Namely, the studies of

T-lymphocyte development in mouse fetal thymic stroma

colonized with immature thymocytes and CD4C cells from

young and old donors have indicated that thymocyte

development is subject to regulation through two CD4C cell

mediated feedback loops. It has been hypothesized that mature

CD4C cells, whether newly generated in the thymus or re-

entrants from the periphery, exert a negative feedback on the

DN to DP transition and DP subset growth, and a positive

feedback on the DP to CD4 SP transition (Mehr et al., 1996,

1997).

Moreover, a more pronounced increase in the frequency of

mature CD4-8C SP than in the frequency of mature CD4C8-

SP seems to be in keeping with the observation indicating that

the CD8 T-cell subset is less sensitive than the CD4 T- cell

subset to age-induced alterations (Pawelec et al., 1999).

The present study also demonstrated that the relative

number of CD4C25C cells was significantly diminished in

the thymus of aged rats reflecting, at least partly, a reduced

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589588

frequency of intrathymically developing Treg cells. (Fontenot

and Rudensky, 2005). Taking collectively the data suggesting

that Thy-1- mediated down-regulation of TCR signalling is

impaired in aged rats and those indicating that naturally arising

Treg cell lineage commitment is induced in response to self-

reactive TCRs with an avidity range for self peptide—MHC

somewhere between that required for positive and negative

selection (Fontenot and Rudensky, 2005), a diminished Treg

cell lineage commitment, and therefore a reduced thymic

production of Treg cells in aged rats may be expected. This

assumption is fully consistent with the increased incidence of

autoimmune disorders in old age (Wick et al., 2000; Shanker,

2004).

The present study confirmed that early differentiational

steps within the CD4-8- DN stage of thymocyte development

are affected in rats by ageing, and demonstrated alterations in

TCRab-dependent stages of T-cell development that, at least

partly, may be related to impaired expression of Thy-1

adhesion and signalling molecules on TCRablow thymocytes

entering selection processes. Furthermore, it suggests that

impaired positive selection and/or overexaggerated negative

selection leads to a reduced frequency of positively selected

CD4C8C DP TCRabhigh cells in aged rats, and that these

changes are followed by more efficient differentiation of

CD4C8C DP TCRabhigh cells into mature SP cells exhibiting

increased pre-migrational proliferation. Moreover, the study

indicates that generation of CD4C Treg cells is also impaired in

the thymus of aged rats, thus providing an explanation for the

increased frequency of autoimmune diseases in aged animals.

Acknowledgements

This work is support by the grants 1239 and 145049B from

Ministry of Science and Environmental Protection of the

Republic of Serbia.

References

Agus, B.D., Surh, C.D., Sprent, J., 1991. Reentry of T cells to the adult thymus

is restricted to activated T cells. J. Exp. Med. 173, 1039–1046.

Ales-Martinez, J.E., Alvarez-Mon, M., Merino, F., Bonilla, F., Martinez-A., C.,

Durantez, A., De la Hera, A., 1988. Decreased TCR-CD3CT cell numbers

in healthy aged humans. Evidence that T cell defects are masked by a

reciprocal increase of TCR-CD3-CD2C natural killer cells. Eur.

J. Immunol. 18, 1827–1830.

Almeida, A.R.M., Borghans, J.A.M., Freitas, A.A., 2001. T cell homeostasis:

thymus regeneration and peripheral T cell restoration in mice with a

reduced fraction of competent precursors. J. Exp. Med. 194, 591–599.

Aspinall, R., 1997. Age-associated thymic atrophy in the mouse is due to a

deficiency affecting rearrangement of the TCR during intrathymic T cell

development. J. Immunol. 158, 3037–3045.

Aspinall, R., 2000. Longevity and immune response. Biogerontology 1,

273–278.

Bodey, B., Bodey Jr., B., Siegel, S.E., Kaiser, H.E., 1997. Involution of the

mammalian thymus, one of the heading regulators of ageing. In vivo 11,

421–440.

Brill, S., Ben-Menahem, D., Kukulansky, T., Tal, E., Globerson, A., 1990. Do

peripheral T-lymphocytes reflect splenic T-lymphocyte functions in

individual young and old mice? Aging: Immunol. Infect. Dis. 2, 221–228.

Brown, D.G., Sun, X.-M., Cohen, G., 1993. Dexamethasone-induced apoptosis

involves cleavage of DNA to large fragments prior to internucleosomal

fragmentation. J. Biol. Chem. 268, 3037–3039.

Cantrell, D.A., 2002. Transgenic analysis of thymocyte signal transduction.

Nat. Rev. Immunol. 2, 20–27.

Capri, M., Quaglino, D., Verzella, G., Monti, D., Bonafe, M., Cossarizza, A.,

Troiano, L., Zecca, L., Pasquali-Ronchetti, I., Franceschi, C., 2000. A

cytofluorimetric study of T lymphocyte subsets in rat lymphoid tissues

(thymus, lymph nodes) and peripheral blood: a continuous remodelling

during the first year of life. Exp. Gerontol. 35, 613–625.

Chan, S.H., Cosgrove, D., Waltzinger, C., Benoist, C., Mathis, D., 1993.

Another view of the selective model of thymocyte selection. Cell 73,

225–236.

Cohen, J.J., 1991. Programmed cell death in murine myeloid leukemia cells.

Photochem. Photobiol. 72, 114–120.

Consolini, R., Legitimo, A., Calleri, A., 2000. Distribution of age-related

thymulin titres in normal subjects through the course of life. Clin. Exp.

Immunol. 121, 444–447.

Donskoy, E., Goldschneider, I., 1992. Thymocytopoiesis is maintained by

blood-borne precursors throughout postnatal life. A study in parabiotic

mice. J. Immunol. 148, 1604–1612.

Fabris, N., Mocchegiani, M., Muzzoli, M., Provinciali, M., 1988. Immune-

neuroendocrine interactions during aging. PNEI 1, 5–20.

Fabris, N., Mocchegiani, M., Provinciali, M., 1997. Plasticity of neuroendo-

crine-thymus interactions during aging. Exp. Gerontol. 32, 415–429.

Fontenot, J.D., Rudensky, A.Y., 2005. A well adapted regulatory contrivance:

regulatory T cell development and the forkhead family transcription factor

Foxp3. Nat. Immunol. 6, 331–337.

Foss, D.L., Donskoy, E., Goldschneider, I., 2001. The importation of

hematogenous precursors by the thymus is a gated phenomenon in normal

adult mice. J. Exp. Med. 193, 365–374.

George, A.J.T., Ritter, M.A., 1996. Thymic involution with ageing:

obsolescence or good housekeeping? Immunol. Today 17, 267–269.

Globerson, A., Effros, R.B., 2000. Ageing of lymphocytes and lymphocytes in

the aged. Immunol. Today 21, 515–521.

Godfrey, D.I., Hammond, K.J., Poulton, L.D., Smyth, M.J., Baxter, A.G.,

2000. NKT cells: facts, functions and fallacies. Immunol. Today 21,

573–583.

Grubeck-Loebenstein, B., 1997. Changes in the aging immune system.

Biologicals 25, 205–208.

Guidos, C.J., 1996. Positive selection of CD4C and CD8C cells. Curr. Opin.

Immunol. 8, 225–232.

Hirokawa, K., Kubo, S., Utsuyama, M., Kurashima, C., Sado, T., 1986. Age-

related changes in the potential of bone marrow cells to repopulate the

thymus and splenic T cells in mice. Cell. Immunol. 100, 443–451.

Hirokawa, K., Utsuyama, M., Kasai, M., Kurashima, C., Ishijima, S.,

Zeng, Y.X., 1994. Understanding the mechanism of age-changes of

thymic function to promote T cell differentiation. Immunol. Lett. 40,

267–277.

Hirokawa, K., Utsuyama, M., Kobayashi, S., 1997. Hypothalamic control of

development and ageing of the thymus. Mech. Ageing Dev. 100,

177–185.

Hsu, H.-C., Mountz, J.D., Williams, R.W., Shelton, B.J., Yang, P.-A.,

Matsuki, Y., Xu, X., Dodd, C.H., Li, L., Geiger, H., Zhang, H.-G.,

Zant, G.V., 2002. Age-related change in thymic T-cell development is

associated with genetic loci on mouse chromosomes 1, 3, and 11. Mech.

Ageing Dev. 123, 1145–1158.

Hsu, H.-C., Li, L., Zhang, H.-G., Mountz, J.D., 2005. Genetic regulation of

thymic involution. Mech. Ageing Dev. 126, 87–97.

Hueber, A.O., Bernard, A.M., Battari, C.L., Marguet, D., Massol, P., Foa, C.,

Brun, N., Garsia, S., Steward, C., Pierres, M., He, H.T., 1997. Thymocytes

in Thy-1K/K mice show augmented TCR signaling and impared

differentiation. Curr. Biol. 7, 705–708.

Hunig, T., Wallny, H.J., Hartley, J.K., Lawetzky, A., Tiefenthaler, G., 1989. A

monoclonal antibody to a constant determinant of the rat T cell antigen

receptor that induces T cell activation. J. Exp. Med. 169, 73–86.

Jameson, S.C., Hogquist, K.A., Bevan, M.J., 1995. Positive selection of

thymocytes. Annu. Rev. Immunol. 13, 93–126.

G. Leposavic et al. / Experimental Gerontology 41 (2006) 574–589 589

Kamath, A.B., Nagarkatti, P.S., Nagarkatti, M., 1998. Characterization of

phenotypic alterations induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin on

thymocytes in vivo and its effect on apoptosis. Toxicol. Appl. Pharmacol.

150, 117–124.

Kapasi, A.A., Singhal, P.C., 1999. Aging splenocyte and thymocyte apoptosis

is associated with enhanced expression of p53, Bax, and Caspase-3. Mol.

Cell. Biol. Res. Commun. 1, 78–81.

Kaplan, P.J., Garvey, J.S., 1981. Age-related changes in responsiveness of

various rat tissue lymphocytes to mitogens. Immunol. Lett. 3, 357–363.

Kelley, K.W., Brief, S., Westly, H.J., Novakofski, J., Bechtel, P.J., Simon, J.,

Walker, E.B., 1986. GH3 pituitary adenoma cells can reverse thymic aging

in rats. Proc. Natl Acad. Sci. USA 83, 5663–5667.

Killeen, N., 1997. Thy-1-hiding in full view. Curr. Biol. 7, R774–R777.

Lau, L., Spain, L.M., 2000. Altered ageing-related thymic involution in T cell

receptor transgenic, MHC-deficient, and CD4-deficient mice. Mech.

Ageing Dev. 114, 101–121.

Leposavic, G., Pejcic-Karapetrovic, B., Kosec, D., 2002. Alterations in

thymopoiesis in intact and peripubertally orchidectomized adult rats of

different age. Mech. Ageing Dev. 123, 401–411.

Leposavic, G., Pekic, S., Kosec, D., 2005. Gonadotropin-releasing hormone

agonist administration affects the thymopoiesis in adult female rats

independently on gonadal hormone production. Am. J. Reprod. Immunol.

53, 1–13.

Li, Y.M., Brunke, D.L., Dantzer, R., Kelley, K., 1992. Pituitary epithelial cell

implants reverse the accumulation of CD4–CD8-lymphocytes in thymus

glands of aged rats. Endocrinology 130, 2703–2709.

Lovatt, M., Yang, T.H., Stauss, H.J., Fisher, A.G., Merkenschlager, M., 2000.

Different doses of agonistic ligand drive the maturation of functional CD4

and CD8 T cells from immature precursors. Eur. J. Immunol. 30, 371–381.

Mackall, C.L., Punt, J.A., Morgan, P., Farr, A.G., Gress, R.E., 1998. Thymic

function in young/old chimeras: substantial thymic T cell regenerative

capacity despite irreversible age-associated thymic involution. Eur.

J. Immunol. 28, 1886–1893.

Madden, K.S., Rajan, S.T., Bellinger, D.B., Felten, S.Y., Felten, D.L., 1997.

Age-associated alterations in sympathetic neural interactions within the

immune system. Dev. Comp. Immunol. 21, 479–486.

Mehr, R., Perelson, A.S., Fridkis-Hareli, M., Globerson, A., 1996. Feedback

regulation of T cell development: manifestations in aging. Mech. Ageing

Dev. 91, 195–210.

Mehr, R., Perelson, A.S., Fridkis-Hareli, M., Globerson, A., 1997. Regulatory

feedback pathways in the thymus. Immunol. Today 18, 581–585.

Michie, A.M., Zuniga-Pflucker, J.C., 2002. Regulation of thymocyte

differentiation: pre-TCR signals and b-selection. Semin. Immunol. 14,

311–323.

Mojcik, C.F., Greiner, D.L., Goldschneider, I., 1991. Characterization of RT6-

bearing rat lymphocytes. Developmental relationships of RT6K and

RT6CT cell. Dev. Immunol. 1, 191–201.

Mower Jr., D.A., Peckham, D.W., Illera, V.A., Fishbaugh, J.K., Stunz, L.L.,

Ashman, R.F., 1994. Decreased membrane phospholipid packing and

decreased size precede DNA cleavage in mature mouse B cell apoptosis.

J. Immunol. 152, 4832–4842.

Nieto, M.A., Gonzalez, A., Gambon, F., Dıaz-Espada, F., Lopez-Rivas, A.,

1992. Apoptosis in human thymocytes after treatment with glucocorticoids.

Clin. Exp. Immunol. 88, 341–344.

Okasha, S.A., Ryu, S., Do, Y., McKallip, R.J., Nagarkatti, M., Nagarkatti, P.S.,

2001. Evidence for estradiol-induced apoptosis and dysregulated T cell

maturation in the thymus. Toxicology 163, 49–62.

Pawelec, G., Effros, R.B., Caruso, C., Remarque, E., Barnett, Y., Solana, R.,

1999. T cells and ageing. Front. Biosci. 4, d216–d269.

Pear, W.S., Tu, L., Stein, P.L., 2004. Lineage choices in the developing

thymus: choosing the T and NKT pathways. Curr. Opin. Immunol. 16,

167–173.

Plecas-Solarovic, B., Lalic, L.J., Leposavic, G., 2004. Age-dependent

morphometrical changes in the thymus of male propranol-treated rats.

Ann. Anat. 186, 141–147.

Pongracz, J., Parnell, S., Anderson, G., Jaffrezou, J.-P., Jenkinson, E., 2003.

Con A activates an Akt/PKB dependent survival mechanism to modulate

TCR induced cell death in double positive thymocytes. Mol. Immunol. 39,

1013–1023.

Reid, S., Cross, R., Snow, E.R., 1996. Combined Hoechst 33342 and

merocyanine 540 staining to examine murine B cell cycle stage, viability

and apoptosis. J. Immunol. Methods 192, 43–54.

Ritter, M.A., Boyd, R.L., 1993. Development in the thymus: it takes two to

tango. Immunol. Today 14, 462–469.

Romanyukha, A.A., Alexei, A., Yashin, A.I., 2003. Age related changes in

population of peripheral T cells: towards a model of immunoscenescence.

Mech. Ageing Dev. 124, 433–443.

Seddon, B., Mason, D., 2000. The third function of the thymus. Immunol.

Today 21, 95–99.

Shanker, A., 2004. Is thymus redundant after adulthood. Immunol. Lett. 91,

79–86.

Sharp, A., Kukulansky, T., Globerson, A., 1990. In vitro analysis of age-related

changes in the developmental potential of bone marrow thymocyte

progenitors. Eur. J. Immunol. 12, 2541–2546.

Shevach, E.M., 2002. CD4CCD25C suppressor T cells: more questions than

answers. Nat. Rev. Immunol. 2, 389–400.

Shortman, K., Vremec, D., Egerton, M., 1991. The kinetics of T cell antigen

receptor expression by subgroups of CD4C8C thymocytes: delineation of

CD4C8C32C thymocytes as post-selection intermediates leading to

mature T cells. J. Exp. Med. 173, 323–332.

Thoman, M.L., 1995. The pattern of T-lymphocyte differentiation is altered

during thymic involution. Mech. Ageing Dev. 58, 167–177.

Thoman, M.L., 1997. Early steps in T cell development are affected by ageing.

Cell. Immunol. 178, 117–123.

Tian, Y.-M., Tian, H.-J., Zhang, G.-Y., Dai, Y.-R., 2003. Effects of ginkgo

biloba extracts (EGb 761) on hydroxyl radical—induced thymocyte

apoptosis and on age-related thymic atrophy and peripheral immune

disfunctions in mice. Mech. Ageing Dev. 124, 977–983.

Tsuchida, M., Konishi, M., Jojima, K., Naito, K., Fujikura, Y., Fukumoto, T.,

1994. Analysis of cell surface antigens on glucocorticoid-treated rat

thymocytes with monoclonal antibodies. Immunol. Lett. 39, 209–217.

Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C., 1995. A

novel assay for apoptosis. Flow cytometric detection of phosphatidylserine

expression on early apoptotic cells using fluorescein labelled Annexin V.

J. Immunol. Methods 184, 39–51.

Wajeman-Chao, S.A., Lancaster, S.A., Graf Jr., L.H., Chambers, D.A., 1998.

Mechanism of catecholamine-mediated destabilization of messenger RNA

encoding Thy-1 protein in T-lineage cells. J. Immunol. 161, 4825–4833.

Weibel, E.R., 1979. Stereological Methods: Practical Methods for Biological

Morphometry, vol. 1. Academic Press, London.

Wick, G., Jansen-Durr, P., Berger, P., Blasko, I., Grubeck-Loebenstein, B.,

2000. Disease of ageing. Vaccine 18, 1567–1583.

Williams, A.F., 1989. The structure of Thy-1 antigen. In: Reif, A.E.,

Schlesinger, M. (Eds.), Cell Surface Antigen Thy-1. Marcel Dekker, New

York, pp. 49–69.

Yehuda, A.B., Friedman, G., Wirtheim, E., Abel, L., Globerson, A., 1998.

Checkpoints in thymocytopoiesis in ageing: expression of recombination

activating genes RAG-1 and RAG-2. Mech. Ageing Dev. 192, 239–247.

Yu, S., Abel, L., Globerson, A., 1997. Thymocyte progenitors and T cell

development in ageing. Mech. Ageing Dev. 94, 103–111.

Zamoyska, R., Lovatt, M., 2004. Signalling in T-lymphocyte development:

integration of signalling pathways is the key. Curr. Opin. Immunol. 16,

191–196.