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FAS) on blood lymphocytes☆
Experimental Gerontology 34 (1999) 659 – 673
Age-related changes in the expression of CD95 (APO1/FAS) on blood lymphocytes夞 Marcella Potestioa, Graham Pawelecb, Gabriele Di Lorenzoc, Giuseppina Candorea, Claudia D’Annaa, Francesco Gervasid, Domenico Lioa, Giovanna Tranchidaa, Calogero Carusoa,*, Giuseppina Colonna Romanoa a
Sezione di Patologia Cellulare e Molecolare, Dipartimento di Biopatologia e Metodologie Biomediche dell’Universita` di Palermo, Palermo, Italy b Section for Transplantation Immunology and Immunohaematology, Second Department of Internal Medicine, University Hospital, Tubingen, Germany c Istituto di Medicina interna e Geriatria dell’Universita` di Palermo, Palermo, Italy d Servizio di Patologia clinica ARNAS, Civico Di Cristina Ascoli, Palermo, Italy Received 11 January 1999; received in revised form 16 March 1999; accepted 8 April 1999
Abstract Aging is associated with alterations of the immune system, thought to be related to an increased susceptibility to infectious diseases, and possibly to cancer and autoimmunity in the elderly. In the present paper we report data obtained on freshly collected blood from 148 healthy subjects of different ages (from cord blood to 102 years old). The subjects were divided into seven age classes (cord blood, 3–11 years, 15–39 years, 41– 60 years, 61–74 years, 75– 84 years, 85–102 years) and their lymphocyte subsets and the expression of the apoptosis-related molecule CD95 were evaluated. In respect of lymphocyte subsets, the major differences were found in the cord-blood samples compared with the oldest old groups. In the cord-blood group, the absolute number of all the lymphocyte subsets was enhanced, but in the oldest group, an increase of CD16⫹ lymphocytes was observed, whereas CD19⫹ lymphocytes, which progressively decrease with age, continue to decrease further in the very old. The data show that the expression of CD95 increases until age 74 years, whereas in the oldest old it tends to decrease again. The trend of CD95 expression seems to be related to the change of expression of CD95 on CD4⫹ lymphocytes, because the CD8⫹/CD95⫹ population rose steadily throughout the entire age range. The evaluation of CD95⫹/CD45R0⫹ lymphocytes shows similar results to those observed analyzing CD95 on total lymphocytes. Fur夞 This work was supported by grants from Ministero dell’Universita´ e della Ricerca Scientifica e Tecnologica (60%) to C.C., G.D.L., and G.C.R., from VERUM Foundation to G.P., and from the University of Palermo, International Scientific Cooperation, to C.C. * Corresponding author. Tel.: ⫹39-91-655-5911; fax: ⫹39-91-655-5933. E-mail address: [email protected] (C. Caruso) 0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 4 1 - 8
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thermore, a constant increase of CD95⫹/CD28⫹ and a related decline of CD28⫹ lymphocytes was observed in all age groups. These data suggest that the expression of CD95 on the different subsets of lymphocytes can be considered a good marker for studies of immunosenescence, because it may be predictive of successful aging, and can partially explain the change in lymphocytes subsets in elderly. © 1999 Elsevier Science Inc. All rights reserved. Keywords: CD95; Cytometry; Elderly; Immunosenescence; Lymphocytes
1. Introduction One of the characteristics of aging is immunosenescence, and it has been hypothesized that the decline of the immune system is related to an increased susceptibility to infectious diseases, and possibly cancer and autoimmunity. The relationship between immune functions and disease or mortality risk has been reviewed by Miller (1996) and is controversial, although longitudinal studies (Ferguson et al., 1995) and studies on centenarians (Franceschi et al., 1995) strongly support the hypothesis that some immunological parameters, such us the number, the subset prevalence, and the ability of T lymphocytes to proliferate, should be useful tools for monitoring aging. In fact, changes in lymphocyte subsets and a reduced ability of lymphocytes to be activated in response to mitogenic stimuli and to proliferate are usually reported in old age (Miller, 1996; Ferguson et al., 1995; Candore et al., 1992; Lio et al., 1996). Besides these data, other studies dissect different age-associated changes in the immune response from the molecular (Pawelec et al., 1995, 1999) and cellular points of view: e.g., the decreased ability of CD28 to provide costimulatory signals (Adibzadeh et al., 1995; Engwerda et al., 1996) and the different pattern of cytokine production, that can also be shown by immunization procedures in which T-cell-dependent antibody production is defective (Adkins and Hamilton, 1992; Adkins et al., 1993; Candore et al., 1993; Caruso et al., 1996a; Castle et al., 1997; Clerici et al., 1993; Forsthuber et al., 1996). We have previously reported a reduced proliferation and cytokine production accompanied by enhanced apoptosis and CD95 expression in cultured lymphocytes of aged individuals (66 – 80 vs. 21– 60 years old); we also reported both an enhanced apoptosis and a increased expression of CD95 in lymphocytes just collected, in a preliminary study (Candore et al., 1992; Potestio et al., 1998). In the present study, we focus on some general parameters evaluated during the different phases of life, from birth to very old age (centenarian). In addition to the lymphocyte markers, we studied the expression of the apoptosis-related molecule APO1/Fas (CD95) on lymphocytes in freshly collected blood in different age groups. In agreement with previous preliminary data (Potestio et al., 1998), in this paper we demonstrate that both the density of CD95 expressed on lymphocytes as well as the number of CD95⫹ cells, is progressively increased during the course of aging and that it reaches the highest levels in donors of the sixth through the seventh decades. It then decreases and in the oldest donors (85–102 years old), values are the same as those observed in adult subjects (15– 60 years old). These alterations observed in total lymphocytes are mainly attributable to the change of expression of CD95 on CD4⫹ lymphocytes, whereas the CD8⫹/CD95⫹ population rose steadily throughout the entire age range. Moreover, we report the increase of CD95 on both CD45R0⫹ and CD45R0⫺ T lym-
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phocytes and the increase of CD95⫹/CD28⫹ T cells concomitant with a decline of the number of CD28⫹ T lymphocytes. These results suggest that the modification of the immune system observed in the elderly might be at least in part related to the elimination of cells via apoptotic mechanisms, thus regulating the size of the immune response. Moreover, the altered expression of CD95 with age implies that it must be evaluated in immunosenescence studies.
2. Materials and methods 2.1. Subjects and analysis of leukocytes A total of 130 subjects (age range, 3–102) and 18 cord-blood samples were studied. In each group the number of males and females was approximately the same. Analysis was performed in seven different groups according to the following age ranges: Y Y Y Y Y Y Y
Group Group Group Group Group Group Group
0: 1: 2: 3: 4: 5: 6:
cord blood (18 samples); 3–11 years old (15 samples); 15–39 years old (32 samples); 41– 60 years old (22 samples); 61–74 years old (22 samples); 75– 84 years old (21 samples); 85–102 years old (18 samples).
None of the selected subjects was affected by neoplastic, infectious or autoimmune diseases and was not receiving any drug-influencing immune functions at the time of the study. All of the subject studied were living in their own home. The smoking status was not investigated. Subjects were not selected according to SENIEUR protocol. In fact, the aim of the present study was the identification of markers that, in an unselected healthy population, may give information about immunosenescence. Neonatal cord blood was collected from the umbilical vein immediately after normal delivery at term. Leukocytes were counted according the manufacturer’s instructions, on a Technicon H*1TM (Bayer, Tarrytown, NY, USA), that allowed us to count four populations of stained cells (neutrophils, eosinophils, basophils, and monocytes) and two populations of unstained cells (lymphocytes and large unstained cells) (Bollinger et al., 1987). 2.2. Immunofluorescence analysis In all the freshly collected blood samples we identified the lymphocyte population by using forward and side-angle scatter (FSC, SSC) on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). Samples were treated with fluorochrome-conjugated monoclonal antibodies (mAbs) anti-CD3, CD4, CD8 (Ortho), CD16, CD20, (Pharmingen, Los Angeles, CA, USA) to identify lymphocyte subpopulations. A red-cell lysant (Ortho-mune, Ortho Diagnostic System, Raritan, NJ, USA) was used to eliminate red cells. Forward and side angle scatter were collected as linear signals and all fluorescent emissions were collected on a four-decade logarithmic scale. Spectral overlap of fluorescent signals was minimized by electronic compensation with CALIBRITE beads (Becton Dickinson) before each determination series. All measurements were made with the same
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instrument setting and at least 104 cells were analyzed by using Lysis II software (Becton Dickinson). The apoptosis-related APO1/FAS molecule was identified by fluorochrome conjugated anti-CD95 mAb (Pharmingen) and reported both as percentage of positive cells and as mean fluorescence intensity (MFI) representing the intensity of CD95 expression on the cell membrane. Isotype-matched negative controls were used to determine the background of fluorescence. To evaluate which kind of lymphocytes were involved in the modifications of CD95, we selected a limited number of subjects of each group of age (4 for cord-blood samples and 5 subjects per groups 1– 6), and performed three-color fluorescence analysis by using mAbs anti-CD3 (fluorescein isothiocyanate-conjugated) (Pharmingen), anti-CD4 or CD8 (PE-conjugated) (Pharmingen), and anti-CD95 (Tri-Color conjugate) (Caltag, Burlingame, CA). Gated CD3⫹ lymphocytes were analyzed evaluating CD95 expression on CD4 and CD8 cells. In the same way we performed a three-color analysis to evaluate CD95 expression on CD3⫹/CD28⫹ (PE, Pharmingen) or CD3/⫹CD45R0⫹ (PE, Pharmingen) T-lymphocytes. In this case, we used anti-CD3 (FITC), anti-CD28 or anti-CD45R0 (PE), and anti-CD95 (Tri-) mAbs. 2.3. Statistics Values given as mean ⫾ SE were compared between the different groups by analysis of variance and the Turkey-HSD test with significance level ⬍0.05. All the data in three-color fluorescence were expressed as mean ⫾ SE and analyzed by Kruskal–Wallis test. As no significant differences were observed in each group between males and females, the results were analyzed from pooled data. To evaluate the statistical correlation between cell values and age, linear regression tests were used.
3. Results 3.1. Analysis of blood-cell populations The absolute values of total leukocytes and of each type of white cells were analyzed in the different age groups and, as shown in Table 1, there were no significant differences among the groups studied, with the exception of cord blood, which showed significantly higher levels of each type of white cell evaluated. 3.2. Age-related changes in lymphocyte subpopulations The highest mean absolute values of CD3, CD4, CD8, CD19, and CD16 positive cells were found in cord-blood samples. Analysis of lymphocyte subsets (Figs. 1 A–E) showed an age-related decrease in T- and B-cell subsets. There was a trend toward a decrease of CD3 (r ⫽ ⫺0.55), CD4 (r ⫽ ⫺0.59), CD8 (r ⫽ ⫺0.46), and CD19 (r ⫽ ⫺0.54) lymphocytes. A different behavior was observed in the oldest group of subjects (Group 6, age 85–102); in this age class there was a slight increase of CD3⫹, CD4⫹ lymphocytes compared with Groups 1–5, but this failed to attain statistical significance; in contrast, the increase of CD16⫹ lymphocytes was statistically significant. The CD19⫹ lymphocytes significantly decreased with age.
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Table 1 The absolute counts (⫻ 103/L ⫾ SE) of the different kinds of white cells evaluated in all the groups studied
Groups
Leucocytes
Lymphocytes
Neutrophils
Monocytes
Eosinophils
Basophils
0 1 2 3 4 5 6
16.5 ⫾ 1.08 7.57 ⫾ 0.33 7.46 ⫾ 0.38 6.67 ⫾ 0.32 6.46 ⫾ 0.53 6.20 ⫾ 0.33 6.83 ⫾ 0.44
4.65 ⫾ 0.48 2.39 ⫾ 0.16 2.10 ⫾ 0.12 1.66 ⫾ 0.11 1.52 ⫾ 0.07 1.54 ⫾ 0.15 1.63 ⫾ 0.13
10.2 ⫾ 0.09 4.13 ⫾ 0.33 4.45 ⫾ 0.29 4.29 ⫾ 0.25 3.98 ⫾ 0.36 4.00 ⫾ 0.28 4.40 ⫾ 0.36
1.25 ⫾ 0.08 0.65 ⫾ 0.03 0.44 ⫾ 0.02 0.37 ⫾ 0.02 0.36 ⫾ 0.02 0.37 ⫾ 0.02 0.46 ⫾ 0.02
0.32 ⫾ 0.05 0.29 ⫾ 0.23 0.20 ⫾ 0.02 0.16 ⫾ 0.16 0.11 ⫾ 0.12 0.12 ⫾ 0.02 0.13 ⫾ 0.22
0.16 ⫾ 0.21 0.05 ⫾ 0.06 0.05 ⫾ 0.04 0.06 ⫾ 0.02 0.04 ⫾ 0.02 0.03 ⫾ 0.02 0.03 ⫾ 0.02
Samples were divided into six groups as follows: 0) cord blood; 1) 3–11 years old; 2) 15–39 years old; 3) 41– 60 years old; 4) 61–74 years old; 5) 75– 84 years old; 6) 85–102 years old. For statistical analysis, the Tukey-HSD test was used; the significance values obtained in the different subsets of lymphocytes are shown: only the samples included into the Group 0 (cord blood) show significant differences with other groups (p ⬍ 0.05).
3.3. CD95 expression on freshly collected lymphocytes increases with age In our previous study (Potestio et al., 1998) it was demonstrated that there was an enhanced expression of the apoptosis-related molecule CD95 associated with an increased percentage of apoptotic cells, in both freshly collected blood and in activated lymphocytes from old donors. In the present paper, we extend the analysis of CD95 expression on a larger panel of subjects. Results (on FSC vs. SSC gated lymphocytes) are expressed either as the percentage of CD95⫹ lymphocytes or as MFI. As shown in Fig. 2A–D, the expression of CD95 progressively increases with age with a good statistically significant correlation. Notably, the highest level of CD95 expression is reached in Group 4 (61–74 years) and then it decreases progressively. 3.4. CD95 expression by lymphocytes subsets ex vivo 3.4.1. Different age-related CD95 expression on CD4 and CD8 T lymphocytes As is well known, CD95 expression on T lymphocytes can lead to apoptosis of these cells, if CD95 is ligated, and if the cells are susceptible. To evaluate whether expression of this marker on T-cell subsets can be related to the modification of immune response observed in old subjects, in a representative number of subjects of each age class, the expression of CD95 on CD3⫹ lymphocytes was evaluated on CD4⫹ or CD8⫹ subsets. The results (Fig. 3A and B) show that the trend in the expression of CD95 on CD4⫹ T lymphocytes is similar to that observed on total lymphocytes (highest levels in the age class 61–74 years old, then decreases again). In contrast, the expression of CD95 on CD8⫹ lymphocytes continues to increase with age and does not show reduction in the oldest age groups. It is therefore concluded that the age-related modifications of the expression of CD95 on lymphocytes is attributable to age-related modifications of the expression of CD95 both on CD4⫹ and CD8⫹ lymphocytes. However, whereas CD95 was reduced again in oldest CD4 cells, it continued to increase on the oldest CD8 cells. 3.4.2. Expression of CD95 on CD45R0⫹ T lymphocytes changes with age Exposure to antigen in vivo leads to modifications of blood T-lymphocyte subsets, in particular the increase of expression of CD45R0⫹ T lymphocytes that reflects the
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Fig. 1. (A–E) The figures show the absolute counts (⫻103/L ⫾ SE) of the different lymphocyte populations. For statistical analysis the Tukey-HSD test was used. Significance was observed between the following groups: (A) Group 0 vs. others groups; Group 1 vs. Groups 3, 4, 5; (B) Group 0 vs. groups 1–5; Group 2 vs. Groups 4, 5; (C) Group 0 vs. others groups; Group 2 vs. Groups 3, 4; (D) Group 0 vs. Groups 1–5; Group 6 vs. Group 4; (E) Group 0 vs. Groups 1–5; Group 6 vs. Groups 1, 2, 3.
acquisition of the memory-preactivated phenotype that increases with age (Cossarizza et al., 1996; Miyawaki et al., 1992). We analyzed CD95 expression on CD45R0⫹ (and CD45R0⫺) T lymphocytes. Fig. 4a shows the percentages of CD45R0⫹ T lymphocytes and the CD45R0⫹/CD95⫹ T lymphocytes evaluated in all the age classes studied. Fig. 4b shows representative results of this kind of experiment, indicating that the age-related increase of CD95 involves both CD45R0⫹ and CD45R0⫺ T lymphocytes. Here again, a decrease of the CD95⫹ population was observed in the oldest subjects. As shown, this alteration is not exclusive to CD45R0⫹ cells as expected considering CD95 a marker of in vivo activation.
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Fig. 2. (A–D) In panels A and C, values of CD95, expressed either as percentage of positive lymphocytes or as MFI, are given as mean ⫾ SE. For statistical analysis the Tukey-HSD test was used. Significance was observed between the following groups: A) Groups 5 and 6 vs. Groups 0, 1, 2; C) Groups 4 and 5 vs. Groups 0, 1, 2. The correlations between age and CD95 expression are depicted: (B) r ⫽ 0.72, p ⬍ 0.0001; (D) r ⫽ 0.63, p ⬍ 0.0001.
3.4.3. Age-related increased expression of CD95 on CD28⫹ T lymphocytes The activation of T cells requires both the stimulation via the T cell receptor (TCR) and the engagement of the costimulatory molecule CD28. It is known that the expression of CD28 is decreased in aging (Effros et al. 1994; Fagnoni et al., 1996) and that this leads to a reduction of T-cell ability to proliferate to mitogenic stimuly (Boucher et al., 1998). Recently, an age-related decrease in rescue from T-cell death after costimulation mediated by CD28 has been observed (Engwerda et al., 1996). Therefore, in a limited number of subjects, we evaluated the expression of CD28 on T lymphocytes and, in the same subjects, the expression of CD95 on CD28⫹ T lymphocytes. The results show that the percentage of CD28⫹ cells changes during the course of aging. There was a reduction of CD28⫹ cells with age, accompanied by an increased percentage of double-positive CD28⫹/CD95⫹ T lymphocytes (Fig. 5). These data suggest that the increasing loss of CD28⫹ cells with age might be related to the increased expression of CD95. Taken together, these data suggest that the expression of CD95 on different peripheral blood T-cell subsets may be considered a sensitive marker of immunosenescence because it may be predictive of successful aging, and might be involved in the elimination of activated immunocompetent cells. 4. Discussion The modifications observed in the immune system of aged subjects are usually designated immunosenescence. To study this phenomenon, different parameters have been
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Fig. 3. The percentages (mean ⫾ SE) of CD3⫹/CD4⫹/CD95⫹ (panel A) and CD3⫹/CD8⫹/CD95⫹ (panel B) lymphocytes are shown as evaluated in the different age classes. The grouping and the number of subjects studied per group are described in Section 2. For statistical analysis, the nonparametric Kruskal–Wallis test was used. Significance was observed between the following groups: (A) Group 0 vs. Groups 4 and 5; 3. (B) No two groups are significantly different at the 0.05 level.
evaluated (Pawelec et al., 1995, 1999; Pawelec and Solana, 1997). In fact, studies on immunological development give even more information about parameters that may be considered useful for this purpose (Cossarizza et al., 1996; Dean, 1997; Miller, 1996; Pawelec et al., 1995, 1999; Pawelec and Solana, 1997). It has been demonstrated that neonatal T lymphocytes and T lymphocytes from old donors produce a superimposable pattern of cytokines (Th2 type), whereas young adults produce essentially Th1 cytokine under mitogenic stimulation (Adkins and Hamilton, 1992; Adkins et al., 1995; Castle et al., 1997; Clerici et al., 1993; Forsthuber et al., 1996). In particular, we have demonstrated that “type 2” cytokine production is modified or increased in the elderly (Caruso et al., 1996a; Lio et al., 1999) whereas ‘type 1’ cytokines are depressed (Caruso et al.,1996a; Lio et al., 1998). Unfortunately, the analysis of cytokine production is expensive and time consuming and so not suitable for routine evaluation of the immune system. In contrast, the evaluation of lymphocytes subsets seems to be a more appropriate tool to obtain information on a large number of subjects. On the other hand, there is no consensus on the modifications of the number of CD3⫹, CD4⫹, and CD8⫹ T lymphocytes in the course of development and of aging and its relationship with survival (reviewed in Miller, 1996; Pawelec et al., 1999). Longitudinal studies demonstrate that the evaluation not of a single parameter, but a cluster of parameters, may give more information about lifespan and
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Fig. 4. (A) The percentage (mean ⫾ SE) of CD45R0⫹ (closed squares) and CD45⫹/CD95⫹ (open squares) are shown for each age group studied. The grouping and the number of subjects studied per group are described in Section 2. For statistical analysis, the nonparametric Kruskal–Wallis test was used. No significant differences were observed between the groups studied. (B) The figure shows representative dot plots of four subjects studied.
suggest that low T-cell proliferation, high CD8, and low CD4 and CD19 values are predictive of reduced survival (Ferguson et al., 1995; Wikby et al., 1998). Results presented, obtained by analyzing blood samples from 148 subjects, show that the absolute numbers of mononuclear and polymorphonuclear leukocytes are not modified during aging, except for cord-blood samples that show significant differences compared with all the other age groups. Similar results were obtained by measuring lymphocyte subsets, except for CD16 and CD19 values. In fact CD19⫹ cells are significantly decreased, whereas CD16 lymphocytes are significantly increased in the oldest group. These results confirm the above-mentioned reports; moreover, the data on CD16 and CD19 seem to be in good agreement with the “immune system remodelling” model in immunosenescence (Franceschi et al., 1995). The authors, in fact, suggest that the term
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Fig. 5. The percentage (mean ⫾ SE) of CD28⫹ (closed squares) and CD28⫹/CD95⫹ (open squares) are shown for each age group studied. The grouping and the number of subjects studied per group are described in Section 2. For statistical analysis, the nonparametric Kruskal–Wallis test was used. No significant differences were observed between the groups studied.
“continuous remodelling” is clearly descriptive of the situation where some immune parameters increase, others decrease and still others remain unchanged. We have previously demonstrated that the expression of CD95 and the percentage of apoptotic cells are increased among cultured lymphocytes from old subjects (Potestio et al., 1998). To evaluate whether the preliminary in vitro observations on CD95 are representative of the in vivo features, we evaluated CD95 expression in different age groups from cord blood to oldest old. The highest level of CD95 expression was observed in the group of 61–74-year-old subjects. It is interesting to note, however, that the very old group 6 (85–102 years) shows a level of CD95 not significantly different from that observed in adulthood (15– 60 years). Similar results were obtained in “spontaneous” apoptosis tests, although no significant correlation was observed between percentage of apoptotic cells and age (unpublished observations). Moreover our findings about the expression of CD95 on lymphocytes of old subjects are in agreement with other findings that report an age-related increase in the expression of Fas at both the protein (Shinohara et al., 1995) and mRNA levels (Aggarwal and Gupta, 1998), although in experimental animals different results have been obtained (Mountz et al., 1997; Wakikawa et al., 1997). We think that it is not possible compare these results with ours due the very different experimental conditions used (i.e., transgenic mice and/or murine splenic cells). Our results seem to suggest an age-related remodulation of CD95 expression; so it would be interesting to study this marker on T-lymphocyte subsets. We evaluated CD95 on CD4⫹ or CD8⫹, or CD28⫹, or CD45R0⫹ T cells. Our results demonstrate that the trend of altered CD95 expression on total lymphocytes is superimposable on the trend of CD3/CD4/CD95⫹ lymphocytes, whereas CD3/CD8/CD95⫹ cells show a different behavior, namely, a progressive increase with age. As described previously (Bradley et al., 1989; Cossarizza et al., 1996; Ernst et al., 1990; Harris et al., 1992), the percentages of CD45R0⫹ (memory/preactivated) and CD45R0⫺ (naive) T lymphocytes are also modified in the course of aging. Many groups have demonstrated that the amount of naive T lymphocytes is high in cord blood and immediately after birth but, thereafter, the marker CD45R0 shows a progressive increase of expression, but a slightly lower percentage in the oldest old individuals. Our present results, confirming the above reported data, demonstrate that the percentage of single
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positive lymphocytes (CD45R0⫹) show, in the different ages, the same modifications described and suggest that the acquired “immunological experience” during growth, determines the switch from the naive to the memory phenotype of T lymphocytes. On the other hand, the evaluation of CD95 on CD45R0⫹ and CD45R0⫺ T-cell populations demonstrates that CD95 is expressed on both CD45R0⫹ and CD45R0⫺ with the same trend observed in the total lymphocyte populations. We may speculate on the biological significance of the different pattern of expression of CD95 on lymphocytes in aging. There is no doubt that CD95 can, under appropriate circumstances, trigger apoptosis on activated cells in vivo (Lynch et al., 1995) and that in cord-blood and neonatal blood samples there is a predominance of lymphocytes bearing the surface molecule CD45RA that identifies “naive/resting” cells, whereas in adulthood, there is a shift toward a CD45R0 phenotype that identifies “memory/preactivated” cells. On the other hand, it has been reported that CD95 is preferentially, but not exclusively, expressed on CD45R0⫹ T lymphocytes (Miyawaki et al., 1992). Therefore, it is not surprising that in cord blood, low expression of CD95 was found, as already reported in neonatal T lymphocytes (Miyawaki et al., 1992). It has also been demonstrated (Cossarizza et al., 1996) that the percentage of CD45R0⫹ increases rapidly between 1 month and 20 years of life with the major change before the fourth decade of life. Our data demonstrate that in spite of the same trend of CD45R0⫹ and CD95/CD45R0⫹ lymphocytes, the two classes of markers are not identical, and as shown on the plot in Fig. 4, in aged individuals, we observe a difference of the expression of CD95 also in the CD45R0⫺ population. Moreover, it is interesting to note that Miyawaki et al. (1992) also demonstrated that CD95 is expressed on T and B lymphocytes, but it is not expressed on natural killer (NK) cells. This means that the use of CD95 and the number of NK cells as parameters to evaluate the immunosenescence does not give us information about the same cells. Another point to evaluate is the consequence of the enhanced expression of CD95 on lymphocytes of old subjects. It is known that during an immune response the activation of apoptosis prevents an excessive clonal expansion; moreover defects in the Fas gene are related to the development of autoimmune diseases (Moutz et al., 1995, 1996). The increased expression of Fas antigen on lymphocytes might suggest an enhanced proneness to undergo apoptosis. This is confirmed by our previous observations (Potestio et al., 1998), as well as other papers (Aggarwal and Gupta 1998, Phelouzat et al., 1997) published during the preparation of this report, which demonstrate that the CD95 in the elderly is fully active. So it can be hypothesized that although there is a large amount of memory-preactivated lymphocytes in the elderly, the well known (Powers, 1994) inability to evoke a strong secondary response to vaccines may be attributable to enhanced apoptosis of “old” cells in comparison with “young” cells. Lymphocytes from patients with active systemic lupus erythematosus express more Fas than lymphocytes from controls and show an enhanced in vitro apoptosis (Emlen et al., 1994; Ohsako et al., 1994). These observations fit well with the data (Candore et al., 1997) that the production of autoantibodies is a characteristic of both systemic lupus erythematosus and senescence. With reference to this, it was demonstrated that in course of the apoptotic process a selective cleavage of autoantigens that can activate the immune system may occur (Casiano et al., 1996; Casciola–Rosen et al., 1995). As already described (Iwai et al., 1994), Fas antigen is constitutively expressed on both neutrophils and monocytes and the level of expression is quite high when compared with the expression on lymphocytes. Our recent data (Di Lorenzo et al., 1999) on the expression of CD95 on neutrophils from our subject sample, show that, in the elderly, there are
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no remarkable changes in MFI, although we observe a slight decrease (data not shown). These changes in the expression may not be relevant because the level of expression is so high at all ages. The same was true for monocytes as well (unpublished observations). It is therefore unlikely that these differences of expression explain the possible differences in the resolution of inflammatory responses of old versus adult or young subjects. Furthermore, results obtained on monocytes and neutrophils, support the concept that the differential expression of CD95 on lymphocytes in aging depends on the immunological history of the lymphocytes, that is obviously different from the history of the other type of blood cells. In fact, whereas monocytes and polymorphonuclear cells are short-lived cells, lymphocytes have the longest half-life among leukocytes and the size and the type of immune response is the result of environmental and genetic factors (Caruso et al., 1996b; Modica et al., 1993; Stassi et al., 1997). On the other hand, these results suggest to us that in old age there are no modifications in the regulation at transcriptional or post-transcriptional levels of Fas protein. Thus, change in the expression of CD95 in aging, is a feature of lymphocytes. The activation of T cells requires costimulatory signals, and it is known that CD28 represents an important costimulatory molecule the expression of which decreases with age (Adibzadeh et al., 1995; Effros et al., 1994; Engwerda et al., 1996). Our preliminary results are in good agreement with these findings, because, as shown in Fig. 5. CD28⫹ T cells decrease mainly in the last decades of life. In addition, evaluation of CD95⫹/CD28⫹ T lymphocytes shows an enhancement of expression of CD95 on CD28⫹ lymphocytes with age. This result may be suggestive of an involvement of CD95 in the age-related depletion of CD28⫹ T lymphocytes that needs to be confirmed in a larger number of subjects. This was not a longitudinal study, so the correlation of the modified expression of CD95 either with longevity, morbidity and mortality, or with morbility, and lifespan (increased incidence of infectious diseases), cannot be assessed, but results obtained in the oldest old group, in which a decrease of CD4/CD95 positive cells, and a decrease of CD45R0⫹/CD95⫹ cells as well as CD45R0⫹ lymphocytes is observed, may suggest that an increased percentage of these lymphocytes is associated with detrimental effects on survival. In contrast, this is not the case with CD8⫹ lymphocyte, because CD8/CD95⫹ lymphocytes, but not the total CD8 lymphocytes increase with age. Taken together these results support the hypothesis that very old people may represent a selected population that, as described, does not escape the aging process (Sansoni et al., 1993), but shows a slower immunosenescence (Cossarizza et al., 1996). In this context we propose to use CD95 expression as a marker for studies of immunosenescence. Increased expression of CD95 on CD8 cells can be considered a neutral marker of age, but not of senescence, whereas an increase of CD4/CD95⫹ lymphocytes or/and CD45R0/CD95⫹ cells could be considered a marker of senescence and its decrease could be considered a marker of successful aging.
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Age-related changes in the expression of CD95 (APO1/FAS) on blood lymphocytes夞 Marcella Potestioa, Graham Pawelecb, Gabriele Di Lorenzoc, Giuseppina Candorea, Claudia D’Annaa, Francesco Gervasid, Domenico Lioa, Giovanna Tranchidaa, Calogero Carusoa,*, Giuseppina Colonna Romanoa a
Sezione di Patologia Cellulare e Molecolare, Dipartimento di Biopatologia e Metodologie Biomediche dell’Universita` di Palermo, Palermo, Italy b Section for Transplantation Immunology and Immunohaematology, Second Department of Internal Medicine, University Hospital, Tubingen, Germany c Istituto di Medicina interna e Geriatria dell’Universita` di Palermo, Palermo, Italy d Servizio di Patologia clinica ARNAS, Civico Di Cristina Ascoli, Palermo, Italy Received 11 January 1999; received in revised form 16 March 1999; accepted 8 April 1999
Abstract Aging is associated with alterations of the immune system, thought to be related to an increased susceptibility to infectious diseases, and possibly to cancer and autoimmunity in the elderly. In the present paper we report data obtained on freshly collected blood from 148 healthy subjects of different ages (from cord blood to 102 years old). The subjects were divided into seven age classes (cord blood, 3–11 years, 15–39 years, 41– 60 years, 61–74 years, 75– 84 years, 85–102 years) and their lymphocyte subsets and the expression of the apoptosis-related molecule CD95 were evaluated. In respect of lymphocyte subsets, the major differences were found in the cord-blood samples compared with the oldest old groups. In the cord-blood group, the absolute number of all the lymphocyte subsets was enhanced, but in the oldest group, an increase of CD16⫹ lymphocytes was observed, whereas CD19⫹ lymphocytes, which progressively decrease with age, continue to decrease further in the very old. The data show that the expression of CD95 increases until age 74 years, whereas in the oldest old it tends to decrease again. The trend of CD95 expression seems to be related to the change of expression of CD95 on CD4⫹ lymphocytes, because the CD8⫹/CD95⫹ population rose steadily throughout the entire age range. The evaluation of CD95⫹/CD45R0⫹ lymphocytes shows similar results to those observed analyzing CD95 on total lymphocytes. Fur夞 This work was supported by grants from Ministero dell’Universita´ e della Ricerca Scientifica e Tecnologica (60%) to C.C., G.D.L., and G.C.R., from VERUM Foundation to G.P., and from the University of Palermo, International Scientific Cooperation, to C.C. * Corresponding author. Tel.: ⫹39-91-655-5911; fax: ⫹39-91-655-5933. E-mail address: [email protected] (C. Caruso) 0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 4 1 - 8
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thermore, a constant increase of CD95⫹/CD28⫹ and a related decline of CD28⫹ lymphocytes was observed in all age groups. These data suggest that the expression of CD95 on the different subsets of lymphocytes can be considered a good marker for studies of immunosenescence, because it may be predictive of successful aging, and can partially explain the change in lymphocytes subsets in elderly. © 1999 Elsevier Science Inc. All rights reserved. Keywords: CD95; Cytometry; Elderly; Immunosenescence; Lymphocytes
1. Introduction One of the characteristics of aging is immunosenescence, and it has been hypothesized that the decline of the immune system is related to an increased susceptibility to infectious diseases, and possibly cancer and autoimmunity. The relationship between immune functions and disease or mortality risk has been reviewed by Miller (1996) and is controversial, although longitudinal studies (Ferguson et al., 1995) and studies on centenarians (Franceschi et al., 1995) strongly support the hypothesis that some immunological parameters, such us the number, the subset prevalence, and the ability of T lymphocytes to proliferate, should be useful tools for monitoring aging. In fact, changes in lymphocyte subsets and a reduced ability of lymphocytes to be activated in response to mitogenic stimuli and to proliferate are usually reported in old age (Miller, 1996; Ferguson et al., 1995; Candore et al., 1992; Lio et al., 1996). Besides these data, other studies dissect different age-associated changes in the immune response from the molecular (Pawelec et al., 1995, 1999) and cellular points of view: e.g., the decreased ability of CD28 to provide costimulatory signals (Adibzadeh et al., 1995; Engwerda et al., 1996) and the different pattern of cytokine production, that can also be shown by immunization procedures in which T-cell-dependent antibody production is defective (Adkins and Hamilton, 1992; Adkins et al., 1993; Candore et al., 1993; Caruso et al., 1996a; Castle et al., 1997; Clerici et al., 1993; Forsthuber et al., 1996). We have previously reported a reduced proliferation and cytokine production accompanied by enhanced apoptosis and CD95 expression in cultured lymphocytes of aged individuals (66 – 80 vs. 21– 60 years old); we also reported both an enhanced apoptosis and a increased expression of CD95 in lymphocytes just collected, in a preliminary study (Candore et al., 1992; Potestio et al., 1998). In the present study, we focus on some general parameters evaluated during the different phases of life, from birth to very old age (centenarian). In addition to the lymphocyte markers, we studied the expression of the apoptosis-related molecule APO1/Fas (CD95) on lymphocytes in freshly collected blood in different age groups. In agreement with previous preliminary data (Potestio et al., 1998), in this paper we demonstrate that both the density of CD95 expressed on lymphocytes as well as the number of CD95⫹ cells, is progressively increased during the course of aging and that it reaches the highest levels in donors of the sixth through the seventh decades. It then decreases and in the oldest donors (85–102 years old), values are the same as those observed in adult subjects (15– 60 years old). These alterations observed in total lymphocytes are mainly attributable to the change of expression of CD95 on CD4⫹ lymphocytes, whereas the CD8⫹/CD95⫹ population rose steadily throughout the entire age range. Moreover, we report the increase of CD95 on both CD45R0⫹ and CD45R0⫺ T lym-
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phocytes and the increase of CD95⫹/CD28⫹ T cells concomitant with a decline of the number of CD28⫹ T lymphocytes. These results suggest that the modification of the immune system observed in the elderly might be at least in part related to the elimination of cells via apoptotic mechanisms, thus regulating the size of the immune response. Moreover, the altered expression of CD95 with age implies that it must be evaluated in immunosenescence studies.
2. Materials and methods 2.1. Subjects and analysis of leukocytes A total of 130 subjects (age range, 3–102) and 18 cord-blood samples were studied. In each group the number of males and females was approximately the same. Analysis was performed in seven different groups according to the following age ranges: Y Y Y Y Y Y Y
Group Group Group Group Group Group Group
0: 1: 2: 3: 4: 5: 6:
cord blood (18 samples); 3–11 years old (15 samples); 15–39 years old (32 samples); 41– 60 years old (22 samples); 61–74 years old (22 samples); 75– 84 years old (21 samples); 85–102 years old (18 samples).
None of the selected subjects was affected by neoplastic, infectious or autoimmune diseases and was not receiving any drug-influencing immune functions at the time of the study. All of the subject studied were living in their own home. The smoking status was not investigated. Subjects were not selected according to SENIEUR protocol. In fact, the aim of the present study was the identification of markers that, in an unselected healthy population, may give information about immunosenescence. Neonatal cord blood was collected from the umbilical vein immediately after normal delivery at term. Leukocytes were counted according the manufacturer’s instructions, on a Technicon H*1TM (Bayer, Tarrytown, NY, USA), that allowed us to count four populations of stained cells (neutrophils, eosinophils, basophils, and monocytes) and two populations of unstained cells (lymphocytes and large unstained cells) (Bollinger et al., 1987). 2.2. Immunofluorescence analysis In all the freshly collected blood samples we identified the lymphocyte population by using forward and side-angle scatter (FSC, SSC) on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). Samples were treated with fluorochrome-conjugated monoclonal antibodies (mAbs) anti-CD3, CD4, CD8 (Ortho), CD16, CD20, (Pharmingen, Los Angeles, CA, USA) to identify lymphocyte subpopulations. A red-cell lysant (Ortho-mune, Ortho Diagnostic System, Raritan, NJ, USA) was used to eliminate red cells. Forward and side angle scatter were collected as linear signals and all fluorescent emissions were collected on a four-decade logarithmic scale. Spectral overlap of fluorescent signals was minimized by electronic compensation with CALIBRITE beads (Becton Dickinson) before each determination series. All measurements were made with the same
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instrument setting and at least 104 cells were analyzed by using Lysis II software (Becton Dickinson). The apoptosis-related APO1/FAS molecule was identified by fluorochrome conjugated anti-CD95 mAb (Pharmingen) and reported both as percentage of positive cells and as mean fluorescence intensity (MFI) representing the intensity of CD95 expression on the cell membrane. Isotype-matched negative controls were used to determine the background of fluorescence. To evaluate which kind of lymphocytes were involved in the modifications of CD95, we selected a limited number of subjects of each group of age (4 for cord-blood samples and 5 subjects per groups 1– 6), and performed three-color fluorescence analysis by using mAbs anti-CD3 (fluorescein isothiocyanate-conjugated) (Pharmingen), anti-CD4 or CD8 (PE-conjugated) (Pharmingen), and anti-CD95 (Tri-Color conjugate) (Caltag, Burlingame, CA). Gated CD3⫹ lymphocytes were analyzed evaluating CD95 expression on CD4 and CD8 cells. In the same way we performed a three-color analysis to evaluate CD95 expression on CD3⫹/CD28⫹ (PE, Pharmingen) or CD3/⫹CD45R0⫹ (PE, Pharmingen) T-lymphocytes. In this case, we used anti-CD3 (FITC), anti-CD28 or anti-CD45R0 (PE), and anti-CD95 (Tri-) mAbs. 2.3. Statistics Values given as mean ⫾ SE were compared between the different groups by analysis of variance and the Turkey-HSD test with significance level ⬍0.05. All the data in three-color fluorescence were expressed as mean ⫾ SE and analyzed by Kruskal–Wallis test. As no significant differences were observed in each group between males and females, the results were analyzed from pooled data. To evaluate the statistical correlation between cell values and age, linear regression tests were used.
3. Results 3.1. Analysis of blood-cell populations The absolute values of total leukocytes and of each type of white cells were analyzed in the different age groups and, as shown in Table 1, there were no significant differences among the groups studied, with the exception of cord blood, which showed significantly higher levels of each type of white cell evaluated. 3.2. Age-related changes in lymphocyte subpopulations The highest mean absolute values of CD3, CD4, CD8, CD19, and CD16 positive cells were found in cord-blood samples. Analysis of lymphocyte subsets (Figs. 1 A–E) showed an age-related decrease in T- and B-cell subsets. There was a trend toward a decrease of CD3 (r ⫽ ⫺0.55), CD4 (r ⫽ ⫺0.59), CD8 (r ⫽ ⫺0.46), and CD19 (r ⫽ ⫺0.54) lymphocytes. A different behavior was observed in the oldest group of subjects (Group 6, age 85–102); in this age class there was a slight increase of CD3⫹, CD4⫹ lymphocytes compared with Groups 1–5, but this failed to attain statistical significance; in contrast, the increase of CD16⫹ lymphocytes was statistically significant. The CD19⫹ lymphocytes significantly decreased with age.
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Table 1 The absolute counts (⫻ 103/L ⫾ SE) of the different kinds of white cells evaluated in all the groups studied
Groups
Leucocytes
Lymphocytes
Neutrophils
Monocytes
Eosinophils
Basophils
0 1 2 3 4 5 6
16.5 ⫾ 1.08 7.57 ⫾ 0.33 7.46 ⫾ 0.38 6.67 ⫾ 0.32 6.46 ⫾ 0.53 6.20 ⫾ 0.33 6.83 ⫾ 0.44
4.65 ⫾ 0.48 2.39 ⫾ 0.16 2.10 ⫾ 0.12 1.66 ⫾ 0.11 1.52 ⫾ 0.07 1.54 ⫾ 0.15 1.63 ⫾ 0.13
10.2 ⫾ 0.09 4.13 ⫾ 0.33 4.45 ⫾ 0.29 4.29 ⫾ 0.25 3.98 ⫾ 0.36 4.00 ⫾ 0.28 4.40 ⫾ 0.36
1.25 ⫾ 0.08 0.65 ⫾ 0.03 0.44 ⫾ 0.02 0.37 ⫾ 0.02 0.36 ⫾ 0.02 0.37 ⫾ 0.02 0.46 ⫾ 0.02
0.32 ⫾ 0.05 0.29 ⫾ 0.23 0.20 ⫾ 0.02 0.16 ⫾ 0.16 0.11 ⫾ 0.12 0.12 ⫾ 0.02 0.13 ⫾ 0.22
0.16 ⫾ 0.21 0.05 ⫾ 0.06 0.05 ⫾ 0.04 0.06 ⫾ 0.02 0.04 ⫾ 0.02 0.03 ⫾ 0.02 0.03 ⫾ 0.02
Samples were divided into six groups as follows: 0) cord blood; 1) 3–11 years old; 2) 15–39 years old; 3) 41– 60 years old; 4) 61–74 years old; 5) 75– 84 years old; 6) 85–102 years old. For statistical analysis, the Tukey-HSD test was used; the significance values obtained in the different subsets of lymphocytes are shown: only the samples included into the Group 0 (cord blood) show significant differences with other groups (p ⬍ 0.05).
3.3. CD95 expression on freshly collected lymphocytes increases with age In our previous study (Potestio et al., 1998) it was demonstrated that there was an enhanced expression of the apoptosis-related molecule CD95 associated with an increased percentage of apoptotic cells, in both freshly collected blood and in activated lymphocytes from old donors. In the present paper, we extend the analysis of CD95 expression on a larger panel of subjects. Results (on FSC vs. SSC gated lymphocytes) are expressed either as the percentage of CD95⫹ lymphocytes or as MFI. As shown in Fig. 2A–D, the expression of CD95 progressively increases with age with a good statistically significant correlation. Notably, the highest level of CD95 expression is reached in Group 4 (61–74 years) and then it decreases progressively. 3.4. CD95 expression by lymphocytes subsets ex vivo 3.4.1. Different age-related CD95 expression on CD4 and CD8 T lymphocytes As is well known, CD95 expression on T lymphocytes can lead to apoptosis of these cells, if CD95 is ligated, and if the cells are susceptible. To evaluate whether expression of this marker on T-cell subsets can be related to the modification of immune response observed in old subjects, in a representative number of subjects of each age class, the expression of CD95 on CD3⫹ lymphocytes was evaluated on CD4⫹ or CD8⫹ subsets. The results (Fig. 3A and B) show that the trend in the expression of CD95 on CD4⫹ T lymphocytes is similar to that observed on total lymphocytes (highest levels in the age class 61–74 years old, then decreases again). In contrast, the expression of CD95 on CD8⫹ lymphocytes continues to increase with age and does not show reduction in the oldest age groups. It is therefore concluded that the age-related modifications of the expression of CD95 on lymphocytes is attributable to age-related modifications of the expression of CD95 both on CD4⫹ and CD8⫹ lymphocytes. However, whereas CD95 was reduced again in oldest CD4 cells, it continued to increase on the oldest CD8 cells. 3.4.2. Expression of CD95 on CD45R0⫹ T lymphocytes changes with age Exposure to antigen in vivo leads to modifications of blood T-lymphocyte subsets, in particular the increase of expression of CD45R0⫹ T lymphocytes that reflects the
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Fig. 1. (A–E) The figures show the absolute counts (⫻103/L ⫾ SE) of the different lymphocyte populations. For statistical analysis the Tukey-HSD test was used. Significance was observed between the following groups: (A) Group 0 vs. others groups; Group 1 vs. Groups 3, 4, 5; (B) Group 0 vs. groups 1–5; Group 2 vs. Groups 4, 5; (C) Group 0 vs. others groups; Group 2 vs. Groups 3, 4; (D) Group 0 vs. Groups 1–5; Group 6 vs. Group 4; (E) Group 0 vs. Groups 1–5; Group 6 vs. Groups 1, 2, 3.
acquisition of the memory-preactivated phenotype that increases with age (Cossarizza et al., 1996; Miyawaki et al., 1992). We analyzed CD95 expression on CD45R0⫹ (and CD45R0⫺) T lymphocytes. Fig. 4a shows the percentages of CD45R0⫹ T lymphocytes and the CD45R0⫹/CD95⫹ T lymphocytes evaluated in all the age classes studied. Fig. 4b shows representative results of this kind of experiment, indicating that the age-related increase of CD95 involves both CD45R0⫹ and CD45R0⫺ T lymphocytes. Here again, a decrease of the CD95⫹ population was observed in the oldest subjects. As shown, this alteration is not exclusive to CD45R0⫹ cells as expected considering CD95 a marker of in vivo activation.
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Fig. 2. (A–D) In panels A and C, values of CD95, expressed either as percentage of positive lymphocytes or as MFI, are given as mean ⫾ SE. For statistical analysis the Tukey-HSD test was used. Significance was observed between the following groups: A) Groups 5 and 6 vs. Groups 0, 1, 2; C) Groups 4 and 5 vs. Groups 0, 1, 2. The correlations between age and CD95 expression are depicted: (B) r ⫽ 0.72, p ⬍ 0.0001; (D) r ⫽ 0.63, p ⬍ 0.0001.
3.4.3. Age-related increased expression of CD95 on CD28⫹ T lymphocytes The activation of T cells requires both the stimulation via the T cell receptor (TCR) and the engagement of the costimulatory molecule CD28. It is known that the expression of CD28 is decreased in aging (Effros et al. 1994; Fagnoni et al., 1996) and that this leads to a reduction of T-cell ability to proliferate to mitogenic stimuly (Boucher et al., 1998). Recently, an age-related decrease in rescue from T-cell death after costimulation mediated by CD28 has been observed (Engwerda et al., 1996). Therefore, in a limited number of subjects, we evaluated the expression of CD28 on T lymphocytes and, in the same subjects, the expression of CD95 on CD28⫹ T lymphocytes. The results show that the percentage of CD28⫹ cells changes during the course of aging. There was a reduction of CD28⫹ cells with age, accompanied by an increased percentage of double-positive CD28⫹/CD95⫹ T lymphocytes (Fig. 5). These data suggest that the increasing loss of CD28⫹ cells with age might be related to the increased expression of CD95. Taken together, these data suggest that the expression of CD95 on different peripheral blood T-cell subsets may be considered a sensitive marker of immunosenescence because it may be predictive of successful aging, and might be involved in the elimination of activated immunocompetent cells. 4. Discussion The modifications observed in the immune system of aged subjects are usually designated immunosenescence. To study this phenomenon, different parameters have been
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Fig. 3. The percentages (mean ⫾ SE) of CD3⫹/CD4⫹/CD95⫹ (panel A) and CD3⫹/CD8⫹/CD95⫹ (panel B) lymphocytes are shown as evaluated in the different age classes. The grouping and the number of subjects studied per group are described in Section 2. For statistical analysis, the nonparametric Kruskal–Wallis test was used. Significance was observed between the following groups: (A) Group 0 vs. Groups 4 and 5; 3. (B) No two groups are significantly different at the 0.05 level.
evaluated (Pawelec et al., 1995, 1999; Pawelec and Solana, 1997). In fact, studies on immunological development give even more information about parameters that may be considered useful for this purpose (Cossarizza et al., 1996; Dean, 1997; Miller, 1996; Pawelec et al., 1995, 1999; Pawelec and Solana, 1997). It has been demonstrated that neonatal T lymphocytes and T lymphocytes from old donors produce a superimposable pattern of cytokines (Th2 type), whereas young adults produce essentially Th1 cytokine under mitogenic stimulation (Adkins and Hamilton, 1992; Adkins et al., 1995; Castle et al., 1997; Clerici et al., 1993; Forsthuber et al., 1996). In particular, we have demonstrated that “type 2” cytokine production is modified or increased in the elderly (Caruso et al., 1996a; Lio et al., 1999) whereas ‘type 1’ cytokines are depressed (Caruso et al.,1996a; Lio et al., 1998). Unfortunately, the analysis of cytokine production is expensive and time consuming and so not suitable for routine evaluation of the immune system. In contrast, the evaluation of lymphocytes subsets seems to be a more appropriate tool to obtain information on a large number of subjects. On the other hand, there is no consensus on the modifications of the number of CD3⫹, CD4⫹, and CD8⫹ T lymphocytes in the course of development and of aging and its relationship with survival (reviewed in Miller, 1996; Pawelec et al., 1999). Longitudinal studies demonstrate that the evaluation not of a single parameter, but a cluster of parameters, may give more information about lifespan and
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Fig. 4. (A) The percentage (mean ⫾ SE) of CD45R0⫹ (closed squares) and CD45⫹/CD95⫹ (open squares) are shown for each age group studied. The grouping and the number of subjects studied per group are described in Section 2. For statistical analysis, the nonparametric Kruskal–Wallis test was used. No significant differences were observed between the groups studied. (B) The figure shows representative dot plots of four subjects studied.
suggest that low T-cell proliferation, high CD8, and low CD4 and CD19 values are predictive of reduced survival (Ferguson et al., 1995; Wikby et al., 1998). Results presented, obtained by analyzing blood samples from 148 subjects, show that the absolute numbers of mononuclear and polymorphonuclear leukocytes are not modified during aging, except for cord-blood samples that show significant differences compared with all the other age groups. Similar results were obtained by measuring lymphocyte subsets, except for CD16 and CD19 values. In fact CD19⫹ cells are significantly decreased, whereas CD16 lymphocytes are significantly increased in the oldest group. These results confirm the above-mentioned reports; moreover, the data on CD16 and CD19 seem to be in good agreement with the “immune system remodelling” model in immunosenescence (Franceschi et al., 1995). The authors, in fact, suggest that the term
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Fig. 5. The percentage (mean ⫾ SE) of CD28⫹ (closed squares) and CD28⫹/CD95⫹ (open squares) are shown for each age group studied. The grouping and the number of subjects studied per group are described in Section 2. For statistical analysis, the nonparametric Kruskal–Wallis test was used. No significant differences were observed between the groups studied.
“continuous remodelling” is clearly descriptive of the situation where some immune parameters increase, others decrease and still others remain unchanged. We have previously demonstrated that the expression of CD95 and the percentage of apoptotic cells are increased among cultured lymphocytes from old subjects (Potestio et al., 1998). To evaluate whether the preliminary in vitro observations on CD95 are representative of the in vivo features, we evaluated CD95 expression in different age groups from cord blood to oldest old. The highest level of CD95 expression was observed in the group of 61–74-year-old subjects. It is interesting to note, however, that the very old group 6 (85–102 years) shows a level of CD95 not significantly different from that observed in adulthood (15– 60 years). Similar results were obtained in “spontaneous” apoptosis tests, although no significant correlation was observed between percentage of apoptotic cells and age (unpublished observations). Moreover our findings about the expression of CD95 on lymphocytes of old subjects are in agreement with other findings that report an age-related increase in the expression of Fas at both the protein (Shinohara et al., 1995) and mRNA levels (Aggarwal and Gupta, 1998), although in experimental animals different results have been obtained (Mountz et al., 1997; Wakikawa et al., 1997). We think that it is not possible compare these results with ours due the very different experimental conditions used (i.e., transgenic mice and/or murine splenic cells). Our results seem to suggest an age-related remodulation of CD95 expression; so it would be interesting to study this marker on T-lymphocyte subsets. We evaluated CD95 on CD4⫹ or CD8⫹, or CD28⫹, or CD45R0⫹ T cells. Our results demonstrate that the trend of altered CD95 expression on total lymphocytes is superimposable on the trend of CD3/CD4/CD95⫹ lymphocytes, whereas CD3/CD8/CD95⫹ cells show a different behavior, namely, a progressive increase with age. As described previously (Bradley et al., 1989; Cossarizza et al., 1996; Ernst et al., 1990; Harris et al., 1992), the percentages of CD45R0⫹ (memory/preactivated) and CD45R0⫺ (naive) T lymphocytes are also modified in the course of aging. Many groups have demonstrated that the amount of naive T lymphocytes is high in cord blood and immediately after birth but, thereafter, the marker CD45R0 shows a progressive increase of expression, but a slightly lower percentage in the oldest old individuals. Our present results, confirming the above reported data, demonstrate that the percentage of single
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positive lymphocytes (CD45R0⫹) show, in the different ages, the same modifications described and suggest that the acquired “immunological experience” during growth, determines the switch from the naive to the memory phenotype of T lymphocytes. On the other hand, the evaluation of CD95 on CD45R0⫹ and CD45R0⫺ T-cell populations demonstrates that CD95 is expressed on both CD45R0⫹ and CD45R0⫺ with the same trend observed in the total lymphocyte populations. We may speculate on the biological significance of the different pattern of expression of CD95 on lymphocytes in aging. There is no doubt that CD95 can, under appropriate circumstances, trigger apoptosis on activated cells in vivo (Lynch et al., 1995) and that in cord-blood and neonatal blood samples there is a predominance of lymphocytes bearing the surface molecule CD45RA that identifies “naive/resting” cells, whereas in adulthood, there is a shift toward a CD45R0 phenotype that identifies “memory/preactivated” cells. On the other hand, it has been reported that CD95 is preferentially, but not exclusively, expressed on CD45R0⫹ T lymphocytes (Miyawaki et al., 1992). Therefore, it is not surprising that in cord blood, low expression of CD95 was found, as already reported in neonatal T lymphocytes (Miyawaki et al., 1992). It has also been demonstrated (Cossarizza et al., 1996) that the percentage of CD45R0⫹ increases rapidly between 1 month and 20 years of life with the major change before the fourth decade of life. Our data demonstrate that in spite of the same trend of CD45R0⫹ and CD95/CD45R0⫹ lymphocytes, the two classes of markers are not identical, and as shown on the plot in Fig. 4, in aged individuals, we observe a difference of the expression of CD95 also in the CD45R0⫺ population. Moreover, it is interesting to note that Miyawaki et al. (1992) also demonstrated that CD95 is expressed on T and B lymphocytes, but it is not expressed on natural killer (NK) cells. This means that the use of CD95 and the number of NK cells as parameters to evaluate the immunosenescence does not give us information about the same cells. Another point to evaluate is the consequence of the enhanced expression of CD95 on lymphocytes of old subjects. It is known that during an immune response the activation of apoptosis prevents an excessive clonal expansion; moreover defects in the Fas gene are related to the development of autoimmune diseases (Moutz et al., 1995, 1996). The increased expression of Fas antigen on lymphocytes might suggest an enhanced proneness to undergo apoptosis. This is confirmed by our previous observations (Potestio et al., 1998), as well as other papers (Aggarwal and Gupta 1998, Phelouzat et al., 1997) published during the preparation of this report, which demonstrate that the CD95 in the elderly is fully active. So it can be hypothesized that although there is a large amount of memory-preactivated lymphocytes in the elderly, the well known (Powers, 1994) inability to evoke a strong secondary response to vaccines may be attributable to enhanced apoptosis of “old” cells in comparison with “young” cells. Lymphocytes from patients with active systemic lupus erythematosus express more Fas than lymphocytes from controls and show an enhanced in vitro apoptosis (Emlen et al., 1994; Ohsako et al., 1994). These observations fit well with the data (Candore et al., 1997) that the production of autoantibodies is a characteristic of both systemic lupus erythematosus and senescence. With reference to this, it was demonstrated that in course of the apoptotic process a selective cleavage of autoantigens that can activate the immune system may occur (Casiano et al., 1996; Casciola–Rosen et al., 1995). As already described (Iwai et al., 1994), Fas antigen is constitutively expressed on both neutrophils and monocytes and the level of expression is quite high when compared with the expression on lymphocytes. Our recent data (Di Lorenzo et al., 1999) on the expression of CD95 on neutrophils from our subject sample, show that, in the elderly, there are
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no remarkable changes in MFI, although we observe a slight decrease (data not shown). These changes in the expression may not be relevant because the level of expression is so high at all ages. The same was true for monocytes as well (unpublished observations). It is therefore unlikely that these differences of expression explain the possible differences in the resolution of inflammatory responses of old versus adult or young subjects. Furthermore, results obtained on monocytes and neutrophils, support the concept that the differential expression of CD95 on lymphocytes in aging depends on the immunological history of the lymphocytes, that is obviously different from the history of the other type of blood cells. In fact, whereas monocytes and polymorphonuclear cells are short-lived cells, lymphocytes have the longest half-life among leukocytes and the size and the type of immune response is the result of environmental and genetic factors (Caruso et al., 1996b; Modica et al., 1993; Stassi et al., 1997). On the other hand, these results suggest to us that in old age there are no modifications in the regulation at transcriptional or post-transcriptional levels of Fas protein. Thus, change in the expression of CD95 in aging, is a feature of lymphocytes. The activation of T cells requires costimulatory signals, and it is known that CD28 represents an important costimulatory molecule the expression of which decreases with age (Adibzadeh et al., 1995; Effros et al., 1994; Engwerda et al., 1996). Our preliminary results are in good agreement with these findings, because, as shown in Fig. 5. CD28⫹ T cells decrease mainly in the last decades of life. In addition, evaluation of CD95⫹/CD28⫹ T lymphocytes shows an enhancement of expression of CD95 on CD28⫹ lymphocytes with age. This result may be suggestive of an involvement of CD95 in the age-related depletion of CD28⫹ T lymphocytes that needs to be confirmed in a larger number of subjects. This was not a longitudinal study, so the correlation of the modified expression of CD95 either with longevity, morbidity and mortality, or with morbility, and lifespan (increased incidence of infectious diseases), cannot be assessed, but results obtained in the oldest old group, in which a decrease of CD4/CD95 positive cells, and a decrease of CD45R0⫹/CD95⫹ cells as well as CD45R0⫹ lymphocytes is observed, may suggest that an increased percentage of these lymphocytes is associated with detrimental effects on survival. In contrast, this is not the case with CD8⫹ lymphocyte, because CD8/CD95⫹ lymphocytes, but not the total CD8 lymphocytes increase with age. Taken together these results support the hypothesis that very old people may represent a selected population that, as described, does not escape the aging process (Sansoni et al., 1993), but shows a slower immunosenescence (Cossarizza et al., 1996). In this context we propose to use CD95 expression as a marker for studies of immunosenescence. Increased expression of CD95 on CD8 cells can be considered a neutral marker of age, but not of senescence, whereas an increase of CD4/CD95⫹ lymphocytes or/and CD45R0/CD95⫹ cells could be considered a marker of senescence and its decrease could be considered a marker of successful aging.
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