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Susceptibility to apoptosis of T lymphocytes from elderly humans is associated with increased in vivo expression of functional Fas receptors
Mechanisms of Ageing and Development 96 (1997) 35 – 46
Susceptibility to apoptosis of T lymphocytes from elderly humans is associated with increased in vivo expression of functional Fas receptors Marie-Anne Phelouzat, Te´re`se Laforge, Agne`s Arbogast, Rafael A. Quadri, Ste´phane Boutet, Jacques J. Proust * Laboratory of Immunobiology of Aging, Department of Geriatrics, Uni6ersity of Gene6a, Route de Mon-Ide´e, 1226 Gene6a-Thoˆnex, Switzerland Received 12 September 1996; received in revised form 1 November 1996; accepted 29 January 1997
Abstract We recently showed that mature T lymphocytes derived from elderly humans were more susceptible to activation-induced cell death than similar cells from young individuals. Because this excessive apoptosis is unrelated to either the age-associated decrease in IL-2 production, a differential Bcl-2 expression or to a modification of the antioxidant pathway, we examined the possibility that the Fas receptor (FasR) is directly implicated in the generation of the unwarranted death signal. We investigated the expression and the function of FasR on T lymphocyte populations from healthy young and elderly individuals. We found that the frequency of FasR + T cells increases as a function of age. The FasR expressed at the surface of freshly isolated T lymphocytes from elderly donors appear to be fully functional since their ligation by a cytocidal IgM anti-Fas mAb leads to a significant increase in DNA fragmentation in this cell population. Conversely, exposure of T cells derived from aged individuals to an antagonistic anti-FasR mAb partially prevents the age-related increase in apoptotic cell death. The population of FasR + T lymphocytes is essentially constituted of previously activated CD45RO + cells and also includes recently activated lymphocytes bearing the CD25 and CD69 activation markers. The accumulation of chronically and recently in vivo activated T-cells with age probably contributes to the amplification of the process of Fas-mediated cell death in T lymphocytes isolated from senescent organisms. © 1997 Elsevier Science Ireland Ltd.
* Corresponding author. Fax: + 41 22 3056525. 0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 1 8 8 3 - 6
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Keywords: Human T lymphocytes; Aging; Cell activation; Fas receptor; Apoptosis
1. Introduction In the immune system of senescent organisms, a number of age-related biochemical alterations have been described that are likely to trigger the apoptotic pathway [1]. For instance, transmembrane signalling defects [2–4], differential expression of genes implicated in the control of the cell cycle [5–7], diminished activity of transcription factors involved in the regulation of lymphokine gene expression [8], poor IL-2 production [9], defective IL-2 receptor expression [9,10] and inappropriate activation of the sphingomyelin-ceramide pathway [11], may all lead to untimely and/or excessive cell death and thus participate in the dysfunction of the immune system classically observed with aging. In previous studies, we demonstrated that increased susceptibility to apoptosis was indeed a characteristic property of mature T lymphocytes derived from elderly humans [12]. In this cellular model, excessive apoptotic cell death appeared unrelated to either the age-associated decrease in IL-2 production, a differential Bcl-2 expression or to a modification of the intracellular content of antioxidant molecules such as glutathione. In an attempt to explore further the molecular mechanisms underlying the age-related increase in T cell apoptosis, we examined the possibility that this excessive T cell death is mediated by the Fas receptor (FasR). FasR (APO-1, CD95) is a cell surface molecule of the tumour necrosis factor and nerve growth factor receptor family [13] able to transduce an apoptotic signal when cross-linked with either appropriate antibodies [14], cells expressing Fas ligand (FasL) [15] or purified FasL [16]. In T lymphocytes, the expression of FasR and its ligand is highly regulated during the process of cellular activation [17]. Depending on the state of activation of the T lymphocytes and the coordinate activity of molecules such as Bcl-2 and related proteins that modulate cell death [18], ligation of FasR may either enhance or terminate the immune response by signalling for the selective elimination of activated cells [19]. In the present investigation, we show that, by comparison with T cells derived from young controls, a significantly higher proportion of T lymphocytes freshly isolated from elderly donors spontaneously express FasR. These in vivo expressed receptors appear to be fully functional since ligation by specific cytocidal antibodies rapidly leads to cell death. Conversely, exposure of T cells derived from aged individuals to blocking anti-FasR mAb partially prevents the age-related increase in apoptotic cell death. In both age groups, essentially all the FasR + cells can be phenotypically characterized as (CD45RO+ ) memory T cells. Additionally, some of them also bear the CD25 and the CD69 activation markers. During the course of aging, the progressive increase in the number of chronically or recently in vivo activated cells may result in excessive susceptibility to apoptosis through a Fasbased mechanism of cell death.
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2. Materials and methods
2.1. Antibodies Supernatant from OKT3 cell line (ATCC, Rockville, MD) was used as a source of anti-CD3 mAb. IgG1 anti-FasR (clone UB2), IgM anti-FasR (clone CH-11) and IgG1 anti-FasR (clone ZB-4) mAbs were purchased from Immunotech (Basel, Switzerland). Phycoerythrin (PE)-conjugated anti-CD45RO, anti-CD45RA, antiCD4, and anti-CD8 were obtained from Sigma (Buchs, Switzerland). PE-conjugated anti-CD25 and anti-CD69 were from Becton-Dickinson (Basel, Switzerland). Goat anti-mouse Ig polyclonal secondary antibodies (unconjugated or conjugated to either FITC or PE) and mouse IgG and IgM isotype control mAbs were obtained from Dako (Zug, Switzerland).
2.2. Cell preparation Heparinized blood was obtained from young (less than 35 years old) donors and from volunteers of over 80 years of age who met the admission criteria for immunogerontological studies (SENIEUR protocol) [20]. For some experiments, volunteers of intermediate age groups were also selected according to the same criteria. Mononuclear cells were separated by centrifugation on a Ficoll-Hypaque cushion (Pharmacia, Duebendorf, Switzerland) and depleted twice from adherent cells by a 1 h incubation, at 37°C, in complete medium (RPMI 1640 containing 10% FCS, 25 mM Hepes and 100 mg/ml gentamycin). This technique yielded cell preparations that were consistently greater than 85% CD3 + , as quantitated by flow cytometry.
2.3. Cell acti6ation, apoptosis induction and apoptosis inhibition After isolation, T lymphocytes were activated by anti-CD3 mAb immobilized on a solid matrix. Briefly, 24 well culture plates (Costar, Dottikon, Switzerland) were first coated with goat anti-mouse Ig as described [21], then exposed to anti-CD3 mAb containing supernatant from OKT3 hybridoma grown in protein free medium (Gibco, Basel, Switzerland). T cells were adjusted at 106/ml in complete medium and were cultured for the indicated time, at 37°C, in the anti-CD3 coated plates. For the induction of apoptosis, 100 ng/ml of cytolytic IgM anti-Fas mAb [22] or the isotype control mAb were added at the initiation of the culture. Cultures were continued for an additional 24 h after which cells were processed for quantitative determination of fragmented cytoplasmic DNA. For inhibition of apoptosis, T cells (at 106/ml) were first incubated for 1 h in the presence of 500 ng/ml of blocking anti-Fas mAb [23] or the control mAb. This treatment was followed by a 24 h activation on anti-CD3 coated plates, in the continued presence of the same concentration of blocking anti-Fas mAb. The cells were then lyzed for quantitation of apoptotic DNA.
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2.4. Quantitati6e determination of apoptotic cell death The quantitative determination of cytoplasmic histone-associated DNA fragments released after apoptosis was performed using the Cell Death Detection ELISA kit (Boehringer Mannheim) following the instructions provided by the manufacturer. Quantitative measurement of cytoplasmic DNA was performed on 2.5 ×105 viable T lymphocytes, as determined microscopically by their ability to exclude the trypan blue.
2.5. Flow cytometric analysis For staining with anti-Fas mAb, T lymphocytes were first incubated with anti-Fas IgG1 mAb (clone UB2), washed and treated with FITC-labelled goat anti-mouse IgG antibodies. For two-colour immunofluorescence analyses, antiFas stained cells were additionally treated with PE-labelled antibodies (either anti-CD4, anti-CD8, anti-CD45R0, anti-CD45RA, anti-CD25 or anti-CD69). All incubations were performed at 4°C, for 30 min, in PBS containing 0.1% BSA/ 0.1% glucose/0.1% sodium azide and followed by two washes in the same medium. Flow cytometry was performed using a FACScan cytofluorograph equipped with Lysys 2 software (Becton-Dickinson) and data were collected on 104 cells. Quadrants and thresholds were set following analysis of cells incubated with isotype-matched control antibodies.
2.6. Data analysis Each experiment was performed simultaneously on paired samples derived from young and elderly subjects. The effect of age was assessed by a pairwise comparison of the observed values using the Wilcoxon signed-rank test.
3. Results
3.1. FasR expression increases as a function of age in freshly isolated human T lymphocytes To address the possible role of FasR in the age-related increase in apoptosis of human T lymphocytes [12], we first examined the spontaneous expression of this surface molecule in T lymphocytes freshly obtained from healthy individuals of different age groups. As shown in Fig. 1A, FasR-related fluorescence intensity displayed by T lymphocytes increases progressively with the age of the donor. Only 16% of T cells derived from the young controls (less than 35 years old) express FasR as compared to 28% in the individuals over 80 years of age (Fig. 1B).
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3.2. Cellular acti6ation upregulates FasR expression independently from age To investigate whether T lymphocytes from the aged differentially upregulate FasR expression, FACS analysis was performed on T lymphocytes that had been activated in vitro by immobilized anti-CD3. Fig. 2 shows the kinetics of FasR expression on T cells stimulated for 24, 48 and 72 h. As shown above, unstimulated cells from the elderly already contain more FasR + T lymphocytes than do cell populations isolated from younger subjects (28 versus 16%). Whereas a significantly higher percentage of FasR + cells is still observed in the 24 h-activated cultures from the elderly (78 as compared to 65% in the control group), no age-related difference can be demonstrated beyond these time points. Following a 72 h activation, over 85% of T lymphocytes from both age groups express FasR (Fig. 2).
3.3. The Fas receptors expressed on the surface of freshly isolated lymphocytes are fully functional To determine whether the spontaneously expressed FasR are able to transduce a death signal, cells were exposed to optimal concentrations (100 ng/ml) of apoptosisinducing anti-FasR mAb for 24 h or to the same amount of control mAb. Since a greater percentage of freshly isolated T lymphocytes from the elderly donors
Fig. 1. Flow cytometric analysis of FasR expression as a function of age. T lymphocytes freshly isolated from the peripheral blood of young and elderly donors were stained with anti-FasR mAb and examined by flow cytometry. A. FasR-related fluorescence intensity of T lymphocytes derived from donors of different age. B. Frequency of FasR + cells in young (less than 35 years old) and aged (over 80 years old) individuals. Each point represents the value corresponding to one subject and the horizontal bar indicates the median for each set of individual values.
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Fig. 2. Kinetics of FasR expression induced by anti-CD3 activation. T lymphocytes from young and aged donors were activated by immobilized anti-CD3 for 24, 48 and 72 h. The cells were then stained with anti-FasR mAb and the percentage of FasR + cells was determined by flow cytometry. Each point represents the value corresponding to one subject and the horizontal bar indicates the median for each set of individual values.
express FasR, ligation of this receptor by the cytocidal IgM-anti-FasR mAb should result in increased cell death in this lymphocyte population. Fig. 3 shows the apoptosis enhancing effect of the anti-FasR IgM mAb in resting and in activated T lymphocytes. Whereas a minimal and comparable amount of histone-associated DNA fragment is measured in the cytoplasmic fraction of freshly isolated T lymphocytes from both age groups, a significantly higher degree of DNA fragmentation is detected in cells derived from the elderly donors after exposure to the anti-FasR mAb (P B0.01). In the absence of cytocidal mAb, anti-CD3 activation results in increased cell death only in T lymphocytes derived from elderly individual. The addition of anti-FasR mAb induces apoptosis in both cell populations. However, anti-CD3 activation-induced cell death is potentiated by anti-FasR mAb to a higher degree (P B0.01) in T lymphocytes from aged individuals. Conversely, the addition of an antagonistic FasR mAb that has been shown to inhibit Fas-mediated apoptosis [23] reduces DNA fragmentation in anti-CD3 activated cells from both age groups. However, in cells from elderly individuals,
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preventing access of a putative ligand to FasR does not allow complete inhibition of the apoptotic process, since the level of fragmentation remains significantly higher than that of unstimulated cells (data not shown).
3.4. Expression of FasR on freshly isolated T lymphocytes correlates with their in 6i6o acti6ated status In order to characterize the T lymphocyte population that accounts for the increase in FasR expression, we first examined FasR distribution in CD4 + and CD8 + T cells by flow cytometry using double labelling. The percentage of FasR + cells in the CD4 + and the CD8 + subpopulations is shown in Table 1. The significantly different percent representation of double positive cells (CD4 + /Fas + or CD8 + /Fas + ) between the two age groups (PB 0.005) reflects the overall increase in the number of FasR + cells with age. However, in both age groups, Fas is preferentially expressed by the CD4 + subpopulation since approximately 75% of the FasR + cells are also CD4 + .
Fig. 3. Apoptosis enhancing effect of cytocidal anti-FasR mAb on T lymphocytes from young and elderly individuals. Unstimulated (®) and anti-CD3 activated T lymphocytes (a-CD3) from donors of both age groups were incubated for 24 h with 100 ng/ml cytocidal anti-Fas IgM mAb (a-Fas) or with the same concentration of control isotype mAb. Cells were then processed for quantitation of fragmented DNA by ELISA as described above. Each point represents the determination of the O.D. value corresponding to one subject and the horizontal bar indicates the median for each set of individual values.
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Table 1 Distribution of Fas+ cells within various T lymphocyte subsets freshly isolated from young and elderly individuals Percent Fas positive cells
Young n Old n
CD4+
CD8+
CD45RO+
CD45RA+
CD25+
CD69+
30.6 23 49.4 23
9.0 23 18.0 23
36.6 20 50.8 20
1.4 20 3.4 20
72.8 18 70.0 18
15.8 10 19.8 10
T lymphocytes from young and elderly donors were first incubated with anti-Fas mAb, treated with FITC-labeled goat anti-mouse antibody. Subsequently these cells were stained with PE-labeled antiCD4, anti-CD8, anti-CD45RO, anti-CD45RA, anti-CD25 or anti-CD69. The percentages of Fas+ cells within each lymphocyte subpopulation were determined by two-colour immunofluorescence analysis.
We next analyzed the expression of FasR with respect to the cellular activation status as assessed by the detection of the activation markers CD45RO, CD45RA, CD25 and CD69. As can be seen in Table 1, the population of CD45RO + T lymphocytes from the elderly group contains significantly more FasR + cells than does the same cell population derived from the young controls (PB 0.005). However, in both age groups, 95.5% of the FasR + cells express the CD45RO phenotype. As usually observed, the percentage of T cells with the memory phenotype (CD45RO + ) is higher in the elderly subjects (59.5%) than in the young ones (48.4%). Although only 20% of the FasR + cells express CD25, in both groups approximately 70% of the CD25 + T lymphocytes are FasR + . By comparison with the cells isolated from young donors, almost twice as many T cells from the elderly express CD69 (19.7 versus 10.7%). However, in both age groups, only a minor fraction of these in vivo activated cells expresses FasR (15.8 in the young versus 19.8% in the aged).
4. Discussion We recently showed that mature activated T lymphocytes derived from elderly humans were more prone to apoptosis than similar cells from young individuals [12]. Among the many mechanisms that may lead to increased activation-induced cell death, we examined the eventuality that FasR, a critical cell surface receptor that mediates apoptotic cell death in lymphoid cells [24], is involved in this age-related phenomenon. In the present study, we investigated the expression and the function of FasR on T lymphocyte populations from healthy young and elderly individuals. We found that the frequency of FasR + cells increases as a function of age. These findings are in agreement with recently published studies that also report an age-related increase in the expression of FasR at both the protein [25] and mRNA levels [26] in human T cells.
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This significant increment of frequency of FasR + T cells with age in humans contrasts with the observations of Zhou et al. who documented an age-associated defect in both the expression and the signalling function of FasR in the murine immune system [27]. Other studies show that anti-CD3-induced apoptosis is considerably greater in cultures derived from aged mice [28,29]. Taken together, these observations suggest that activation-induced cell death of mature murine T lymphocytes occurs through a Fas-independent pathway at a later age. However, other works showed evidence that elimination of activated cells by a Fas-based mechanism predominates in aged mice [30]. In humans, an increase in the frequency of FasR + T lymphocytes resulting in excessive apoptotic cell death has been observed in pathologic conditions such as systemic lupus erythematosus (SLE), autoimmune hepatitis and chronic hepatitis [31,32]. Remarkably, these diseases are characterized by the production of autoantibodies, an anomaly also classically associated with the process of immune senescence. It is therefore conceivable that, in autoimmune diseases as well as in age-related immune dysfunction, the generation of autoantibodies may be related to the presence of FasR + T lymphocytes. Following cellular activation by anti-CD3, T lymphocytes derived from the elderly upregulate FasR to the same extent as cells from the young controls. The kinetics of FasR induction is comparable in both age groups and allows cell cultures from aged individuals to maintain a significantly higher number of FasR + cells up to 24 h after activation. After a 72 h induction, both cell populations contain the same percentage of FasR + lymphocytes. Interestingly, FasR expressed on the surface of T lymphocytes freshly isolated from the elderly donors are fully functional since their ligation by the cytocidal IgM anti-Fas mAb leads to a significant increase in DNA fragmentation in this cell population. These findings are reminiscent of the observations made in patients with active SLE whose freshly isolated FasR + T lymphocytes also showed characteristic DNA fragmentation within 24 h of anti-FasR mAb treatment [33]. The functionality of these receptors is also confirmed by the fact that blocking anti-FasR mAb can partially inhibit the excess of apoptotic cell death in anti-CD3-activated cells from elderly individuals. These results indicate that the apparently spontaneous in vivo expression of FasR is directly implicated in the greater susceptibility of T lymphocytes from aged humans to apoptosis. It is likely that the in vivo expression of the FasR on T cells from elderly donors is not truly constitutive but rather reflects the activated status of these cells in peripheral blood. We therefore attempted to characterize the FasR + T cells with respect to activation, by analysing the expression of various surface molecules known to be markers of early activation (CD69), recent activation (CD25) and chronic activation (CD45RO). In our studies, essentially all the FasR + T lymphocytes (95.5%), irrespective of the age of the donor, belong to the subpopulation of memory (or previously activated) CD45RO + cells. These results are consistent with a previous report [34] which also clearly demonstrated, on lymphocytes freshly isolated from normal mature adults, that FasR was preferentially expressed on CD45RO + populations
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of CD4 + and CD8 + T cells. The different distribution of CD45RO + cells classically observed between young and aged donors partially contributes to the higher percentage of FasR+ cells in the elderly. Although the large majority (over 70%) of recently activated T cells still bearing the a-chain of the IL-2 receptor (CD25) expresses the FasR, this population constitutes a minor proportion (20%) of the FasR + T cell population. The early activated CD69 + T cells are twice as numerous in the elderly as compared to the young adults (19.7 versus 10.7%). However, they contribute marginally to the population of FasR + cells, since a small fraction of them (B 20%) express FasR. The weak correlation between FasR and CD69 may be explained by the fact that the early activation marker CD69 is only transiently expressed following activation [35]. Taken together, these observations suggest that the population of FasR + T lymphocytes is essentially composed of a variety of in vivo stimulated cells with different timing of activation. Aside from the canonical age-associated increase of memory T cells [36], it has been shown that the proportion of recently activated cells is higher in the peripheral blood of aged humans [37]. The accumulation of chronically activated as well as recently activated cells is probably responsible for the increasing number of FasR + T lymphocytes in the course of ageing. Whether the extreme susceptibility to activation-induced cell death of T lymphocytes from the elderly is solely attributable to a greater proportion of FasR + cells among them remains an unanswered question. Although FasR + cell populations from young and aged donors share the same phenotypic characteristics, they may be functionally different with regard to apoptosis. In particular, diverse conditions of activation may result in differential expression of Fas ligand and/or intracellular proteins that modulate cell death, thus leading to distinct sensitivities to apoptosis. It is also conceivable that the threshold of Fas-mediated death signalling is lower in T lymphocytes derived from elderly humans. Death signals generated at the level of FasR are transduced by a sphingomyelin-ceramide pathway [38], the activity of which is spontaneously and considerably elevated during the process of senescence [11]. Under these conditions of constitutive preactivation of the ceramide pathway, even a weak Fas-mediated signal might result in excessive cell death in T lymphocyte populations from senescent organisms.
Acknowledgements This work was supported by grants from the Swiss National Science Foundation (Contract No. 31-33563.92) and the Sandoz Foundation for Gerontological Research.
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[21] L.J. Wysocki and V.L. Sato, ‘Panning’ for lymphocytes: A method for cell selection. Proc. Natl. Acad. Sci. USA, 75 (1978) 2844 – 2848. [22] S. Yonehara, A. Ishii and M. Yonehara, A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor for tumor necrosis factor. J. Exp. Med., 169 (1989) 1747–1756. [23] S. Yonehara, Y. Nishimura, S. Kishii, M. Yonehara, K. Takazawa, T. Tamatani and A. Ishii, Involvement of apoptosis antigen Fas in clonal deletion of human thymocytes. Internat. Immunol., 6 (1994) 1849–1856. [24] R. Watanabe-Fukunaga, C.I. Brannan, N.G. Copeland, N.A. Jenkins and S. Nagata, Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature, 356 (1992) 314–317. [25] S. Shinohara, T. Sawada, Y. Nishioka, S. Tohma, T. Kisaki, T. Inoue, K. Ando, M. Ikeda, H. Fujii and K. Ito, Differential expression of Fas antigen and Bcl-2 protein on CD4 + T cells, CD8 + T cells, and monocytes. Cell. Immunol., 163 (1995) 303 – 308. [26] S. Aggarwal, A. Gupta and S. Gupta, Age-related changes in genes regulating apoptosis in human T cells. Report on the First Int. Conf. on Immunology and Aging, 1996, S-11. [27] T. Zhou, C.K. Edwards III and J.D. Mountz, Prevention of age-related T cell apoptosis defect in CD2-Fas-transgenic mice. J. Exp. Med., 182 (1995) 129 – 137. [28] D.S. Ucker, L.D. Hebshi, J.F. Bloomquist and B.E. Torbett, Physiological T-Cell death: Susceptibility is modulated by activation, aging and transformation, but the mechanism is constant. Immunol. Re6., 142 (1994) 273– 299. [29] F.J. Chrest, M.A. Buchholz, Y.-H. Kim, T.K. Kwon and A.A. Nordin, Anti-CD3-induced apoptosis in T-cells from young and old mice. Cytometry, 20 (1995) 33 – 42. [30] J.C. Rathmell and C.C. Goodnow, Effect of the lpr mutation on elimination and inactivation of self-reactive B cells. J. Immunol., 153 (1994) 2831 – 2842. [31] W. Emlen, J. Niebur and R. Kadera, Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. J. Immunol., 152 (1994) 3685 – 3692. [32] S. Ogawa, K. Sakaguchi, A. Takaki, K. Shiraga, Increased expression of Fas on CD4 + T cells in autoimmune hepatitis and chronic hepatitis type C associated with autoimmune phenomena. Hepatology, 20 (1994) 193. [33] S. Ohsako, M. Hara, M. Harigai, C. Fukasawa and S. Kashiwasaki, Expression and function of Fas antigen and bcl-2 in human systemic lupus erythematosus. Clin. Immunol. Immunopathol., 73 (1994) 109–114. [34] T. Miyawaki, T. Uehara, R. Nibu, T. Tsuji, A. Yachie, S. Yonehara and N. Taniguchi, Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood. J. Immunol., 149 (1992) 3753 – 3758. [35] R. Testi, D. D’Ambrosio, R. De Maria and A. Santoni, The CD69 receptor: A multipurpose cell-surface trigger for hematopoietic cells. Immunol. Today, 15 (1994) 479 – 483. [36] X.N. Xu, I. Beckman, M. Ahern and J. Bradley, A comprehensive analysis of peripheral blood lymphocytes in healthy aged human by flow cytometry. Immun. Cell. Biol., 71 (1993) 549 – 557. [37] M. Utsuyama, K. Hirokawa, C. Kurashima, M. Fukayama, T. Inamatsu, K. Suzuki, W. Hashimoto and K. Sato, Differential age-change in the number of CD4 + CD45RA + and CD4 + CD29 + T cell subsets in human peripheral blood. Mech. Ageing De6., 63 (1992) 57 – 68. [38] B.M. Gill, H. Nishikata, G. Chan, T.L. Delovitch and A. Ochi, Fas antigen and sphingomyelin-ceramide turnover-mediated signalling: Role in life and death of T lymphocytes. Immunol. Re6., 142 (1994) 113–125.
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Susceptibility to apoptosis of T lymphocytes from elderly humans is associated with increased in vivo expression of functional Fas receptors Marie-Anne Phelouzat, Te´re`se Laforge, Agne`s Arbogast, Rafael A. Quadri, Ste´phane Boutet, Jacques J. Proust * Laboratory of Immunobiology of Aging, Department of Geriatrics, Uni6ersity of Gene6a, Route de Mon-Ide´e, 1226 Gene6a-Thoˆnex, Switzerland Received 12 September 1996; received in revised form 1 November 1996; accepted 29 January 1997
Abstract We recently showed that mature T lymphocytes derived from elderly humans were more susceptible to activation-induced cell death than similar cells from young individuals. Because this excessive apoptosis is unrelated to either the age-associated decrease in IL-2 production, a differential Bcl-2 expression or to a modification of the antioxidant pathway, we examined the possibility that the Fas receptor (FasR) is directly implicated in the generation of the unwarranted death signal. We investigated the expression and the function of FasR on T lymphocyte populations from healthy young and elderly individuals. We found that the frequency of FasR + T cells increases as a function of age. The FasR expressed at the surface of freshly isolated T lymphocytes from elderly donors appear to be fully functional since their ligation by a cytocidal IgM anti-Fas mAb leads to a significant increase in DNA fragmentation in this cell population. Conversely, exposure of T cells derived from aged individuals to an antagonistic anti-FasR mAb partially prevents the age-related increase in apoptotic cell death. The population of FasR + T lymphocytes is essentially constituted of previously activated CD45RO + cells and also includes recently activated lymphocytes bearing the CD25 and CD69 activation markers. The accumulation of chronically and recently in vivo activated T-cells with age probably contributes to the amplification of the process of Fas-mediated cell death in T lymphocytes isolated from senescent organisms. © 1997 Elsevier Science Ireland Ltd.
* Corresponding author. Fax: + 41 22 3056525. 0047-6374/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 1 8 8 3 - 6
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Keywords: Human T lymphocytes; Aging; Cell activation; Fas receptor; Apoptosis
1. Introduction In the immune system of senescent organisms, a number of age-related biochemical alterations have been described that are likely to trigger the apoptotic pathway [1]. For instance, transmembrane signalling defects [2–4], differential expression of genes implicated in the control of the cell cycle [5–7], diminished activity of transcription factors involved in the regulation of lymphokine gene expression [8], poor IL-2 production [9], defective IL-2 receptor expression [9,10] and inappropriate activation of the sphingomyelin-ceramide pathway [11], may all lead to untimely and/or excessive cell death and thus participate in the dysfunction of the immune system classically observed with aging. In previous studies, we demonstrated that increased susceptibility to apoptosis was indeed a characteristic property of mature T lymphocytes derived from elderly humans [12]. In this cellular model, excessive apoptotic cell death appeared unrelated to either the age-associated decrease in IL-2 production, a differential Bcl-2 expression or to a modification of the intracellular content of antioxidant molecules such as glutathione. In an attempt to explore further the molecular mechanisms underlying the age-related increase in T cell apoptosis, we examined the possibility that this excessive T cell death is mediated by the Fas receptor (FasR). FasR (APO-1, CD95) is a cell surface molecule of the tumour necrosis factor and nerve growth factor receptor family [13] able to transduce an apoptotic signal when cross-linked with either appropriate antibodies [14], cells expressing Fas ligand (FasL) [15] or purified FasL [16]. In T lymphocytes, the expression of FasR and its ligand is highly regulated during the process of cellular activation [17]. Depending on the state of activation of the T lymphocytes and the coordinate activity of molecules such as Bcl-2 and related proteins that modulate cell death [18], ligation of FasR may either enhance or terminate the immune response by signalling for the selective elimination of activated cells [19]. In the present investigation, we show that, by comparison with T cells derived from young controls, a significantly higher proportion of T lymphocytes freshly isolated from elderly donors spontaneously express FasR. These in vivo expressed receptors appear to be fully functional since ligation by specific cytocidal antibodies rapidly leads to cell death. Conversely, exposure of T cells derived from aged individuals to blocking anti-FasR mAb partially prevents the age-related increase in apoptotic cell death. In both age groups, essentially all the FasR + cells can be phenotypically characterized as (CD45RO+ ) memory T cells. Additionally, some of them also bear the CD25 and the CD69 activation markers. During the course of aging, the progressive increase in the number of chronically or recently in vivo activated cells may result in excessive susceptibility to apoptosis through a Fasbased mechanism of cell death.
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2. Materials and methods
2.1. Antibodies Supernatant from OKT3 cell line (ATCC, Rockville, MD) was used as a source of anti-CD3 mAb. IgG1 anti-FasR (clone UB2), IgM anti-FasR (clone CH-11) and IgG1 anti-FasR (clone ZB-4) mAbs were purchased from Immunotech (Basel, Switzerland). Phycoerythrin (PE)-conjugated anti-CD45RO, anti-CD45RA, antiCD4, and anti-CD8 were obtained from Sigma (Buchs, Switzerland). PE-conjugated anti-CD25 and anti-CD69 were from Becton-Dickinson (Basel, Switzerland). Goat anti-mouse Ig polyclonal secondary antibodies (unconjugated or conjugated to either FITC or PE) and mouse IgG and IgM isotype control mAbs were obtained from Dako (Zug, Switzerland).
2.2. Cell preparation Heparinized blood was obtained from young (less than 35 years old) donors and from volunteers of over 80 years of age who met the admission criteria for immunogerontological studies (SENIEUR protocol) [20]. For some experiments, volunteers of intermediate age groups were also selected according to the same criteria. Mononuclear cells were separated by centrifugation on a Ficoll-Hypaque cushion (Pharmacia, Duebendorf, Switzerland) and depleted twice from adherent cells by a 1 h incubation, at 37°C, in complete medium (RPMI 1640 containing 10% FCS, 25 mM Hepes and 100 mg/ml gentamycin). This technique yielded cell preparations that were consistently greater than 85% CD3 + , as quantitated by flow cytometry.
2.3. Cell acti6ation, apoptosis induction and apoptosis inhibition After isolation, T lymphocytes were activated by anti-CD3 mAb immobilized on a solid matrix. Briefly, 24 well culture plates (Costar, Dottikon, Switzerland) were first coated with goat anti-mouse Ig as described [21], then exposed to anti-CD3 mAb containing supernatant from OKT3 hybridoma grown in protein free medium (Gibco, Basel, Switzerland). T cells were adjusted at 106/ml in complete medium and were cultured for the indicated time, at 37°C, in the anti-CD3 coated plates. For the induction of apoptosis, 100 ng/ml of cytolytic IgM anti-Fas mAb [22] or the isotype control mAb were added at the initiation of the culture. Cultures were continued for an additional 24 h after which cells were processed for quantitative determination of fragmented cytoplasmic DNA. For inhibition of apoptosis, T cells (at 106/ml) were first incubated for 1 h in the presence of 500 ng/ml of blocking anti-Fas mAb [23] or the control mAb. This treatment was followed by a 24 h activation on anti-CD3 coated plates, in the continued presence of the same concentration of blocking anti-Fas mAb. The cells were then lyzed for quantitation of apoptotic DNA.
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2.4. Quantitati6e determination of apoptotic cell death The quantitative determination of cytoplasmic histone-associated DNA fragments released after apoptosis was performed using the Cell Death Detection ELISA kit (Boehringer Mannheim) following the instructions provided by the manufacturer. Quantitative measurement of cytoplasmic DNA was performed on 2.5 ×105 viable T lymphocytes, as determined microscopically by their ability to exclude the trypan blue.
2.5. Flow cytometric analysis For staining with anti-Fas mAb, T lymphocytes were first incubated with anti-Fas IgG1 mAb (clone UB2), washed and treated with FITC-labelled goat anti-mouse IgG antibodies. For two-colour immunofluorescence analyses, antiFas stained cells were additionally treated with PE-labelled antibodies (either anti-CD4, anti-CD8, anti-CD45R0, anti-CD45RA, anti-CD25 or anti-CD69). All incubations were performed at 4°C, for 30 min, in PBS containing 0.1% BSA/ 0.1% glucose/0.1% sodium azide and followed by two washes in the same medium. Flow cytometry was performed using a FACScan cytofluorograph equipped with Lysys 2 software (Becton-Dickinson) and data were collected on 104 cells. Quadrants and thresholds were set following analysis of cells incubated with isotype-matched control antibodies.
2.6. Data analysis Each experiment was performed simultaneously on paired samples derived from young and elderly subjects. The effect of age was assessed by a pairwise comparison of the observed values using the Wilcoxon signed-rank test.
3. Results
3.1. FasR expression increases as a function of age in freshly isolated human T lymphocytes To address the possible role of FasR in the age-related increase in apoptosis of human T lymphocytes [12], we first examined the spontaneous expression of this surface molecule in T lymphocytes freshly obtained from healthy individuals of different age groups. As shown in Fig. 1A, FasR-related fluorescence intensity displayed by T lymphocytes increases progressively with the age of the donor. Only 16% of T cells derived from the young controls (less than 35 years old) express FasR as compared to 28% in the individuals over 80 years of age (Fig. 1B).
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3.2. Cellular acti6ation upregulates FasR expression independently from age To investigate whether T lymphocytes from the aged differentially upregulate FasR expression, FACS analysis was performed on T lymphocytes that had been activated in vitro by immobilized anti-CD3. Fig. 2 shows the kinetics of FasR expression on T cells stimulated for 24, 48 and 72 h. As shown above, unstimulated cells from the elderly already contain more FasR + T lymphocytes than do cell populations isolated from younger subjects (28 versus 16%). Whereas a significantly higher percentage of FasR + cells is still observed in the 24 h-activated cultures from the elderly (78 as compared to 65% in the control group), no age-related difference can be demonstrated beyond these time points. Following a 72 h activation, over 85% of T lymphocytes from both age groups express FasR (Fig. 2).
3.3. The Fas receptors expressed on the surface of freshly isolated lymphocytes are fully functional To determine whether the spontaneously expressed FasR are able to transduce a death signal, cells were exposed to optimal concentrations (100 ng/ml) of apoptosisinducing anti-FasR mAb for 24 h or to the same amount of control mAb. Since a greater percentage of freshly isolated T lymphocytes from the elderly donors
Fig. 1. Flow cytometric analysis of FasR expression as a function of age. T lymphocytes freshly isolated from the peripheral blood of young and elderly donors were stained with anti-FasR mAb and examined by flow cytometry. A. FasR-related fluorescence intensity of T lymphocytes derived from donors of different age. B. Frequency of FasR + cells in young (less than 35 years old) and aged (over 80 years old) individuals. Each point represents the value corresponding to one subject and the horizontal bar indicates the median for each set of individual values.
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Fig. 2. Kinetics of FasR expression induced by anti-CD3 activation. T lymphocytes from young and aged donors were activated by immobilized anti-CD3 for 24, 48 and 72 h. The cells were then stained with anti-FasR mAb and the percentage of FasR + cells was determined by flow cytometry. Each point represents the value corresponding to one subject and the horizontal bar indicates the median for each set of individual values.
express FasR, ligation of this receptor by the cytocidal IgM-anti-FasR mAb should result in increased cell death in this lymphocyte population. Fig. 3 shows the apoptosis enhancing effect of the anti-FasR IgM mAb in resting and in activated T lymphocytes. Whereas a minimal and comparable amount of histone-associated DNA fragment is measured in the cytoplasmic fraction of freshly isolated T lymphocytes from both age groups, a significantly higher degree of DNA fragmentation is detected in cells derived from the elderly donors after exposure to the anti-FasR mAb (P B0.01). In the absence of cytocidal mAb, anti-CD3 activation results in increased cell death only in T lymphocytes derived from elderly individual. The addition of anti-FasR mAb induces apoptosis in both cell populations. However, anti-CD3 activation-induced cell death is potentiated by anti-FasR mAb to a higher degree (P B0.01) in T lymphocytes from aged individuals. Conversely, the addition of an antagonistic FasR mAb that has been shown to inhibit Fas-mediated apoptosis [23] reduces DNA fragmentation in anti-CD3 activated cells from both age groups. However, in cells from elderly individuals,
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preventing access of a putative ligand to FasR does not allow complete inhibition of the apoptotic process, since the level of fragmentation remains significantly higher than that of unstimulated cells (data not shown).
3.4. Expression of FasR on freshly isolated T lymphocytes correlates with their in 6i6o acti6ated status In order to characterize the T lymphocyte population that accounts for the increase in FasR expression, we first examined FasR distribution in CD4 + and CD8 + T cells by flow cytometry using double labelling. The percentage of FasR + cells in the CD4 + and the CD8 + subpopulations is shown in Table 1. The significantly different percent representation of double positive cells (CD4 + /Fas + or CD8 + /Fas + ) between the two age groups (PB 0.005) reflects the overall increase in the number of FasR + cells with age. However, in both age groups, Fas is preferentially expressed by the CD4 + subpopulation since approximately 75% of the FasR + cells are also CD4 + .
Fig. 3. Apoptosis enhancing effect of cytocidal anti-FasR mAb on T lymphocytes from young and elderly individuals. Unstimulated (®) and anti-CD3 activated T lymphocytes (a-CD3) from donors of both age groups were incubated for 24 h with 100 ng/ml cytocidal anti-Fas IgM mAb (a-Fas) or with the same concentration of control isotype mAb. Cells were then processed for quantitation of fragmented DNA by ELISA as described above. Each point represents the determination of the O.D. value corresponding to one subject and the horizontal bar indicates the median for each set of individual values.
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Table 1 Distribution of Fas+ cells within various T lymphocyte subsets freshly isolated from young and elderly individuals Percent Fas positive cells
Young n Old n
CD4+
CD8+
CD45RO+
CD45RA+
CD25+
CD69+
30.6 23 49.4 23
9.0 23 18.0 23
36.6 20 50.8 20
1.4 20 3.4 20
72.8 18 70.0 18
15.8 10 19.8 10
T lymphocytes from young and elderly donors were first incubated with anti-Fas mAb, treated with FITC-labeled goat anti-mouse antibody. Subsequently these cells were stained with PE-labeled antiCD4, anti-CD8, anti-CD45RO, anti-CD45RA, anti-CD25 or anti-CD69. The percentages of Fas+ cells within each lymphocyte subpopulation were determined by two-colour immunofluorescence analysis.
We next analyzed the expression of FasR with respect to the cellular activation status as assessed by the detection of the activation markers CD45RO, CD45RA, CD25 and CD69. As can be seen in Table 1, the population of CD45RO + T lymphocytes from the elderly group contains significantly more FasR + cells than does the same cell population derived from the young controls (PB 0.005). However, in both age groups, 95.5% of the FasR + cells express the CD45RO phenotype. As usually observed, the percentage of T cells with the memory phenotype (CD45RO + ) is higher in the elderly subjects (59.5%) than in the young ones (48.4%). Although only 20% of the FasR + cells express CD25, in both groups approximately 70% of the CD25 + T lymphocytes are FasR + . By comparison with the cells isolated from young donors, almost twice as many T cells from the elderly express CD69 (19.7 versus 10.7%). However, in both age groups, only a minor fraction of these in vivo activated cells expresses FasR (15.8 in the young versus 19.8% in the aged).
4. Discussion We recently showed that mature activated T lymphocytes derived from elderly humans were more prone to apoptosis than similar cells from young individuals [12]. Among the many mechanisms that may lead to increased activation-induced cell death, we examined the eventuality that FasR, a critical cell surface receptor that mediates apoptotic cell death in lymphoid cells [24], is involved in this age-related phenomenon. In the present study, we investigated the expression and the function of FasR on T lymphocyte populations from healthy young and elderly individuals. We found that the frequency of FasR + cells increases as a function of age. These findings are in agreement with recently published studies that also report an age-related increase in the expression of FasR at both the protein [25] and mRNA levels [26] in human T cells.
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This significant increment of frequency of FasR + T cells with age in humans contrasts with the observations of Zhou et al. who documented an age-associated defect in both the expression and the signalling function of FasR in the murine immune system [27]. Other studies show that anti-CD3-induced apoptosis is considerably greater in cultures derived from aged mice [28,29]. Taken together, these observations suggest that activation-induced cell death of mature murine T lymphocytes occurs through a Fas-independent pathway at a later age. However, other works showed evidence that elimination of activated cells by a Fas-based mechanism predominates in aged mice [30]. In humans, an increase in the frequency of FasR + T lymphocytes resulting in excessive apoptotic cell death has been observed in pathologic conditions such as systemic lupus erythematosus (SLE), autoimmune hepatitis and chronic hepatitis [31,32]. Remarkably, these diseases are characterized by the production of autoantibodies, an anomaly also classically associated with the process of immune senescence. It is therefore conceivable that, in autoimmune diseases as well as in age-related immune dysfunction, the generation of autoantibodies may be related to the presence of FasR + T lymphocytes. Following cellular activation by anti-CD3, T lymphocytes derived from the elderly upregulate FasR to the same extent as cells from the young controls. The kinetics of FasR induction is comparable in both age groups and allows cell cultures from aged individuals to maintain a significantly higher number of FasR + cells up to 24 h after activation. After a 72 h induction, both cell populations contain the same percentage of FasR + lymphocytes. Interestingly, FasR expressed on the surface of T lymphocytes freshly isolated from the elderly donors are fully functional since their ligation by the cytocidal IgM anti-Fas mAb leads to a significant increase in DNA fragmentation in this cell population. These findings are reminiscent of the observations made in patients with active SLE whose freshly isolated FasR + T lymphocytes also showed characteristic DNA fragmentation within 24 h of anti-FasR mAb treatment [33]. The functionality of these receptors is also confirmed by the fact that blocking anti-FasR mAb can partially inhibit the excess of apoptotic cell death in anti-CD3-activated cells from elderly individuals. These results indicate that the apparently spontaneous in vivo expression of FasR is directly implicated in the greater susceptibility of T lymphocytes from aged humans to apoptosis. It is likely that the in vivo expression of the FasR on T cells from elderly donors is not truly constitutive but rather reflects the activated status of these cells in peripheral blood. We therefore attempted to characterize the FasR + T cells with respect to activation, by analysing the expression of various surface molecules known to be markers of early activation (CD69), recent activation (CD25) and chronic activation (CD45RO). In our studies, essentially all the FasR + T lymphocytes (95.5%), irrespective of the age of the donor, belong to the subpopulation of memory (or previously activated) CD45RO + cells. These results are consistent with a previous report [34] which also clearly demonstrated, on lymphocytes freshly isolated from normal mature adults, that FasR was preferentially expressed on CD45RO + populations
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of CD4 + and CD8 + T cells. The different distribution of CD45RO + cells classically observed between young and aged donors partially contributes to the higher percentage of FasR+ cells in the elderly. Although the large majority (over 70%) of recently activated T cells still bearing the a-chain of the IL-2 receptor (CD25) expresses the FasR, this population constitutes a minor proportion (20%) of the FasR + T cell population. The early activated CD69 + T cells are twice as numerous in the elderly as compared to the young adults (19.7 versus 10.7%). However, they contribute marginally to the population of FasR + cells, since a small fraction of them (B 20%) express FasR. The weak correlation between FasR and CD69 may be explained by the fact that the early activation marker CD69 is only transiently expressed following activation [35]. Taken together, these observations suggest that the population of FasR + T lymphocytes is essentially composed of a variety of in vivo stimulated cells with different timing of activation. Aside from the canonical age-associated increase of memory T cells [36], it has been shown that the proportion of recently activated cells is higher in the peripheral blood of aged humans [37]. The accumulation of chronically activated as well as recently activated cells is probably responsible for the increasing number of FasR + T lymphocytes in the course of ageing. Whether the extreme susceptibility to activation-induced cell death of T lymphocytes from the elderly is solely attributable to a greater proportion of FasR + cells among them remains an unanswered question. Although FasR + cell populations from young and aged donors share the same phenotypic characteristics, they may be functionally different with regard to apoptosis. In particular, diverse conditions of activation may result in differential expression of Fas ligand and/or intracellular proteins that modulate cell death, thus leading to distinct sensitivities to apoptosis. It is also conceivable that the threshold of Fas-mediated death signalling is lower in T lymphocytes derived from elderly humans. Death signals generated at the level of FasR are transduced by a sphingomyelin-ceramide pathway [38], the activity of which is spontaneously and considerably elevated during the process of senescence [11]. Under these conditions of constitutive preactivation of the ceramide pathway, even a weak Fas-mediated signal might result in excessive cell death in T lymphocyte populations from senescent organisms.
Acknowledgements This work was supported by grants from the Swiss National Science Foundation (Contract No. 31-33563.92) and the Sandoz Foundation for Gerontological Research.
References [1] M.-A. Phelouzat, R.A. Quadri and J.J. Proust, Apoptose et senescence. Me´decine/Sciences, 11 (1995) 894–900.
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