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Age-associated modulation of apoptosis and activation in murine B lymphocytes
Mechanisms of Ageing and Development 103 (1998) 285 – 299
Age-associated modulation of apoptosis and activation in murine B lymphocytes Vongthip Souvannavong a,*, Christophe Lemaire a, Karine Andre´au a, Spencer Brown b, Arlette Adam a a
CNRS-ERS 571, Institut de Biochimie, Baˆt. 430, Uni6ersite´ Paris-Sud, Orsay Cedex 91405, France b CNRS-UPR 40, ISV, Gif sur Y6ette, 91198, France Received 7 February 1998; received in revised form 8 April 1998; accepted 15 April 1998
Abstract We have investigated the influence of age on B-cell responsiveness. The present study showed that the B-cell mitogen, lipopolysaccharide (LPS), similarly stimulated the proliferation of purified B lymphocytes obtained from either young mice (3 months) or old mice (24 months). In contrast, expression of the differentiation marker, alkaline phosphatase (ALP), was about fourfold higher in young mice than in older mice upon stimulation with LPS or with dextran sulfate (DXS) and interleukin-5 (IL-5). The occurrence of apoptosis during aging was then studied: unexpectedly, spontaneous cell death was double in B lymphocytes from young mice compared to older animals. Stimulation with DXS with or without IL-5 rescued B lymphocytes from cell death in young mice but protection decreased with aging, and no longer occurred in 24-month-old mice B cells. Meanwhile, the protective activity conferred by IL-4 was maintained at similar levels throughout aging. However, B cells from old mice were more responsive to apoptosis induction with cycloheximide, dibutyryl cAMP and dexamethasone. Together, the present results indicate an age-associated alteration in apoptosis and activation of B lymphocytes which could contribute to the age-related decline of the immune response. © 1998 Elsevier Science Ireland Ltd. All rights reserved.
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Keywords: Murine B lymphocytes; Aging; Proliferation; Alkaline phosphatase; Apoptosis
1. Introduction It is well recognised that aging is associated with a decline in immune function leading to various infectious and neoplastic diseases in man as well as in experimental animals (Miller, 1991; Chiarugi et al., 1994; Pawelec et al., 1995; Rubin et al., 1996). This age-related deterioration has been attributed to many factors including environmental changes and a failure of senescent cells themselves (Thoman and Weigle, 1989; Hodes, 1995; Proust et al., 1996; Burns and Goodwin, 1997; Hodes, 1997; Warner, 1997). The age-related changes in the antibody response is well documented and has been associated with alterations in T-cell help, in accessory cells and also with intrinsic B-cell defects (Goidl et al., 1976; Burns et al., 1990; Miller and Kelsoe, 1995; Borghesi and Nicoletti, 1996; Miller, 1996; Hodes, 1997; Segre and Segre, 1997). A more limited number of studies has been performed to evaluate the influence of age on the proliferative response of B cells. A decrease of the proliferative response of unseparated spleen cells to B-cell and T-cell mitogens has been reported leading to conflicting interpretations on whether age-related reduction was really due to an intrinsic defect of B cells or to a decrease in the number of responding cells (Abraham et al., 1977; Averill and Wolf, 1985). The proliferative response of purified human peripheral blood B cells to stimulation with anti-IgM or anti-m, with the polyclonal activator SAC, or with mAb against CD20 or CD40, has been reported to be reduced in elderly subjects (Whisler et al., 1991). In contrast, an age-related increase of the proliferative response of B lymphocytes to stimulation with anti-CD40 antibody has been demonstrated in mice in spite of the expression of comparable levels of CD40 in young and in old animals (Song et al., 1997). Paradoxically, while overall immune responsiveness decreases with age, autoimmunity appears to increase (Goidl et al., 1981; Zhao et al., 1995). The age-related qualitative and quantitative changes in antibody production, reflecting alteration in B-cell repertoire and generally associated with deficiency in T-cell help (Song et al., 1997), might also result from a failure of autoreactive cells to undergo cell death (Baixeras et al., 1994; Maclennan, 1995). Apoptosis, considered as physiological cell death as opposed to degenerative cell death (Kerr et al., 1972), plays a fundamental role in the homeostasis of the immune system and is essential in the negative selection and deletion of autoreactive cells (Golstein et al., 1991; Cohen et al., 1992). Many studies have been performed on the T-cell lineage but there is limited information on the role of aging in B-lymphocyte susceptibility to apoptosis. Alkaline phosphatase (ALP, EC 3.1.3.1) belongs to the family of glycosyl-phosphatidylinositol (GPI)-anchored proteins which play an important role in signal
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transduction (Ferguson and Williams, 1988; Anderson, 1993). ALP expression has been associated with B-lymphocyte differentiation (Burg and Feldbush, 1989; Marquez et al., 1990; Souvannavong et al., 1992, 1994) and with the transduction of activation signals (Marty and Feldbush, 1993). ALP was used here as a marker of B-cell differentiation. In the present study, the influence of aging on B-cell apoptosis and activation has been investigated. The results indicated that the proliferative response of purified B lymphocytes was maintained through aging, while their apoptotic susceptibility and capacity for activation were modified.
2. Materials and methods
2.1. Animals Female BDF1 (C57BL/6 ×DBA2)F1 and C3H/HeJ mice were purchased from IFFA CREDO (L’Arbresle, France) and CSAL (La Source, France), respectively, at 8 weeks of age and maintained until use in a sterile microisolator cage system at our central animal care facility.
2.2. Antibodies, interleukins and reagents Recombinant mouse IL-4 and IL-5 were obtained from Genzyme (Cambridge, MA). Monoclonal anti-Thy-1.2, anti-L3T4 (GK1.5), anti-Lyt-2 and anti-Lyt-3 were obtained from Serotec (Oxford, UK). The culture medium used throughout was RPMI 1640 supplemented with 25 mM Hepes, 2 mM L-glutamine, standard antibiotics, 50 mM 2-mercaptoethanol and 8% heat-inactivated fetal calf serum (Gibco, Paisley, Scotland). Cycloheximide (CHX), 6-diamidino-2-phenylindol (DAPI), dexamethasone (DEXA), dextran sulfate (DXS, 500000 MW), dibutyryl cAMP (dbcAMP), dibutyryl cGMP, fluorescein-diacetate (FDA), and lipopolysaccharide (LPS, from Salmonella enteritidis) were purchased from Sigma (St. Louis, MO). RU 486 was a kind gift of Roussel Uclaf, Romainville, France.
2.3. Preparation of purified B cells and culture B lymphocytes were purified as previously described (Souvannavong et al., 1994). Briefly, splenocytes (1 – 2×107/ml in RPMI medium containing 8% FCS) were treated with a cocktail of anti-T-cell monoclonal antibodies at 4°C for 45 min followed by incubation with a 1:10 dilution of low-tox rabbit complement at 37°C for 50 min. Cells were layered on top of a discontinuous Percoll gradient and cells at the 55 – 65% interface were recovered. More than 98% of these cells were found to be surface Ig-positive with FITC-conjugated rabbit anti-mouse Ig antibodies; such purified B cells were unresponsive to Concanavalin A, but retained full responsiveness to LPS. B cells, at 5× 105 cells/ml, were cultured in 24-well plates (Nunclon) in 6% CO2 at 37°C, in medium alone or in the presence of stimuli. These
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Fig. 1. Effect of LPS on the proliferation of splenic and purified B cells. Spleen cells (SC) or purified B cells from old and young BDF1 mice were stimulated with LPS. (A) Kinetics of [3H]TdR incorporation by cells stimulated with LPS (5 mg/ml) for 1 – 4 days; (B) dose-effect of LPS at day 3. (C) Purified B cells from young (shaded bars) and old (solid bars) mice were cultured for 3 days in the presence of LPS (5 mg/ml) with or without dexamethasone (5 nM) and RU 486 (100 nM). Cells (1 ×105/well) were cultured in 96-well culture plates and pulse labeled with 18 kBq/well of [3H]TdR for the final 6 h. Results are expressed as mean cpm9 SD for pooled triplicate cultures from three experiments. Mean values were compared using the Student’s t-test (old versus young mice), differences being significant (*) for P= 0.05, and highly significant (**) for P= 0.001.
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cells were used for gel electrophoresis of DNA or for determination of ALP activity, and cells fixed in 70% ethanol were kept at − 20°C until analysis of DNA content by flow cytometry.
2.4. Cell proliferation Whole splenocytes or purified B cells (1× 105/0.2 ml) were cultured in 96-well plates in medium alone or in the presence of stimuli. Proliferation was assessed at the indicated days by pulsing cultures with 0.5 mCi of [3H]thymidine ([3H]TdR specific activity, 5 Ci/mmol, 1 mCi= 37 kBq, Amersham, Les Ulis, France) 6 h before harvesting. Results are expressed as the mean cpm9S.D. for triplicate cultures.
2.5. ALP assay Cellular alkaline phosphatase activity was determined after 5 days of culture by p-NPP hydrolysis. Briefly, 100 ml of p-NPP dissolved at 1 mg/ml in buffer consisting of 0.1 M diethanolamine, 1 mM MgCl2, and 0.5% Triton X-100, pH 10.5, was added to cell pellets corresponding to 5×104 cells. After a 20-min incubation at 37°C, the reaction was stopped by addition of 50 ml 0.3 N NaOH. Results are reported as the mean arbitrary absorbance (A) units for triplicate wells measured at 405 nm, using a Dynatech microplate reader (MR 700).
2.6. Apoptosis analysis Apoptosis was determined by three methods including a morphological study, a quantitative analysis of apoptotic hypodiploid cells by flow cytometry, and DNA electrophoresis. Cell viability was assessed by FDA staining. Apoptosis was determined morphologically after staining with DAPI. Morphological criteria for apoptotic cell death were chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. DNA fragmentation was assessed by electrophoresis as previously described (Sarih et al., 1993). Briefly, DNA was extracted from lysates of 5 × 105 B cells after treatment with proteinase K and RNAse A. Electrophoresis was performed in agarose gel containing 1 mg/ml ethidium bromide and DNA visualised in UV light. The rate of apoptosis was determined by flow cytometry (Darzynkiewicz et al., 1992). Ethanol-fixed cells (5× 105 cells/ml) were washed twice with HBSS and stained with DAPI (2 mg/ml) at 37°C for 30 min. DNA analysis was then performed on a PARTEC CA II flow cytometer (Chemunex, Maisons-Alfort, France) equipped with a 100-W mercury lamp. Fluorescence at 455 nm was recorded as a function of relative DNA content. Each cytogram was generated till the analysis reached at least 15000 cells. The percentage of apoptotic cells was determined from the sub-G1 peak on cytograms.
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Fig. 2. Effect of age on ALP expression by B lymphocytes. (A) Purified B cells from young (3 months) and old (24 months) normal mice were stimulated with LPS (0.01 – 25 mg/ml). (B) Purified B cells obtained from 2–24-month-old C3H/HeJ mice were cultured in the presence or absence of DXS or DXS+ IL-5 as indicated. ALP activity was determined after 5 days of culture in 5 × 104 cells by p-NPP hydrolysis and expressed as arbitrary absorbance units at 405 nm. Standard deviation bars are given.
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2.7. Statistical analysis Results are expressed as the arithmetic mean9 S.D. for pooled triplicate cultures from three experiments. Mean values were compared by using Student’s t-test for unpaired observations, and the level of significance is indicated in Figs. 1–5, a P-value of 0.05 being considered as significant, and of 0.001 as highly significant.
Fig. 3. Apoptosis of B lymphocytes in young and old mice. Purified B cells from young (3 months) and old (24 months) normal BDF1 mice were cultured for 20 h. (A) Cells were stained with DAPI and analyzed for DNA content; frequency of nuclei with hypodiploid DNA is proportional to the percentage of apoptosis and is used for quantitation. (B) Apoptosis, as demonstrated by laddering of DNA from young (lanes 1–3) and old (lanes 4–6) mice, of B cells cultured in the absence (lanes 1 and 4) or in the presence of CHX (2.5 mg/ml, lanes 2 and 5) or dbcAMP (100 mM, lanes 3 and 6).
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Fig. 4. Age-related effect of various stimuli on B-cell apoptosis. B cells from young (shaded bars) and old (solid bars) normal BDF1 mice (A) and LPS-hyporesponsive C3H/HeJ mice (B) were cultured for 20 h with or without LPS (2.5 mg/ml), dexamethasone (5 nM), DXS (5 mg/ml) and IL-5 (5 units/ml), IL-4 (20 units/ml), CHX (2.5 mg/ml), dbcAMP (100 mM), or dbcGMP (100 mM), as indicated. Quantification of apoptosis was performed by determination of sub-G1 peak (see Fig. 3A). Results are expressed as the percentage of controls. Statistical analysis was performed as described in Fig. 1.
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3. Results
3.1. LPS-induced proliferation is similar in B cells from young and old mice It has been reported that the proliferative response of whole spleen cells to T-cell and B-cell mitogens declined with age (Abraham et al., 1977; Averill and Wolf, 1985). We thus examined whether the response of purified B lymphocytes was similarly affected by aging. Fig. 1A shows that the kinetics of the proliferative response of both spleen cells and B cells to LPS were similar in old and in young mice, with an optimal response between day 2 and day 3. Furthermore Fig. 1B shows that, in a dose-dependent way, LPS increased the proliferation of splenocytes, cell proliferation at the effective dose (1 mg/ml and above) being in young mice (3 months) about double that in old mice (24 months). We then compared the response of purified B cells from young and old mice. The dose-response curve showed that proliferation rates were similar in B cells obtained from either young or old mice. In addition, LPS-induced proliferation was inhibited to a similar extent by the synthetic glucocorticoid, dexamethasone, in young and in old mice. This activity was mediated by glucocorticoid receptors since it was totally inhibited by the antagonist RU 486 (Fig. 1C).
Fig. 5. Influence of various stimuli on apoptosis of B cells from mice of varying ages. B cells were obtained from C3H/HeJ mice of increasing ages (3 – 24 months) and cultured for 20 h with or without DXS, IL-5, IL-4, CHX or dbcAMP. Quantification of apoptosis was determined as described in Fig. 3 and expressed as the percentage of controls. Standard deviation bars are given.
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3.2. ALP production by acti6ated B cells declines with increasing age Expression of ALP is considered as a marker of the differentiation of B lymphocytes. In the present study, expression rate of ALP was determined in LPS-stimulated B cells from both young (3 months) and old (24 months) normal BDF1 mice. Fig. 2 shows that LPS (10 ng–25 mg/ml), dose-dependently increased ALP activity in B cells from young mice, a significant ALP activity being obtained with a dose as low as 100 ng/ml and optimal activity being reached at 2.5 mg/ml LPS and maintained for higher doses. LPS was much less effective at stimulating ALP activity in B cells from old mice, with only a marginal increase whatever the dose used (Fig. 2A). To investigate further this considerable age-associated reduction of ALP expression, we analysed the response of LPS-hyporesponsive C3H/HeJ mice of increasing ages (2 – 24 months). B lymphocytes were treated with the polyclonal B-cell activator, dextran sulfate, and/or IL-5 which have been previously found to stimulate optimal ALP activity (Souvannavong et al., 1992). Results presented in Fig. 2B show that dextran sulfate, in an age-dependent way, stimulated the production of ALP by B cells. Its activity was further potentiated by IL-5 which by itself was unable to trigger ALP expression (data not shown). The capacity of B cells to produce ALP was age-dependent: it was maximal in young adults between 2 and 5 months of age and sharply decreased at 10 months and thereafter. ALP levels in stimulated B cells from old mice were low when compared to those obtained from B cells of young mice, nevertheless, they were higher than background levels of ALP in non-stimulated control cells at any age.
3.3. Age-related changes in spontaneous and drug-modified apoptosis B lymphocytes from young adults (3 months) and from old mice (24 months) were cultured with or without various stimuli known to affect apoptosis. Experiments were performed in parallel in normal BDF1 mice and in LPS-hyporesponsive C3H/HeJ mice. When similar results were obtained for both strains, only those obtained from one strain are presented. The viability of B cells derived from young and old mice was greater than 98% at the initiation of cultures. Apoptosis was evaluated by morphological observation after DNA staining with DAPI (not shown), by flow cytometric analysis of sub-G1 DNA content and by DNA gel electrophoresis. As shown in Fig. 3A, apoptosis spontaneously occurred after 20 h of incubation: cell death appeared to be greater in B cells from young mice (about 35%) than in older mice (about 20%). Apoptosis was accompanied by DNA fragmentation (Fig. 3B), this being higher in young (lane 1) than in old (lane 4) mice. Consistent with previous findings (Illera et al., 1993; Newell et al., 1993), we observed that the translation inhibitor, cycloheximide, and dibutyryl cAMP stimulated apoptosis; moreover, cell death of old mice appeared to be the most affected in spite of lower levels of spontaneous apoptosis (lanes 5 and 6 versus lanes 2 and 3).
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We then investigated whether protection against apoptosis was also affected by aging (Fig. 4). In agreement with previous reports on the repression of apoptosis in B cells (Norvell et al., 1995; Ashman et al., 1996), we observed that dextran sulfate and the cytokine, IL-4, reduced apoptosis of B cells in both mouse strains. As expected, LPS did not affect apoptosis in C3H/HeJ mice (Fig. 4B) but it protected normal B cells in an age-independent way (Fig. 4A). Stimulation with DXS and IL-5 protected young B cells from apoptosis (50 and 30% of controls for BDF1 and C3H/HeJ cells, respectively). The protection by DXS and IL-5 was very low in old normal mice and not significant in old C3H/HeJ mice. In contrast, the protective effect of IL-4 was independent of age in both strains (60 and 50% of controls for C3H/HeJ and BDF1 mice, respectively). On the other hand, stimulation of apoptosis appeared to be higher in B cells of old mice than in those of young mice. Thus, dexamethasone and CHX induced up to fourfold apoptosis in old mice versus twofold in young mice. Similarly, B-cell apoptosis linked to cAMP increase was more notably enhanced in older than in younger mice. An increase of cGMP levels, however, only marginally affected apoptosis whatever the strain or the age of mice.
3.4. Influence of 6arious stimuli on apoptosis of B cells from mice of 6arying ages In order to better define the influence of aging on the susceptibility to apoptosis, we examined the response of B cells from mice of increasing ages (3, 5, 10, 16, and 24 months). B cells were cultured in the presence or absence of various stimuli, and apoptosis was determined by flow cytometry as previously described. The results presented in Fig. 5 show that, independently of age, IL-5 failed to affect apoptosis whereas IL-4 partially rescued B cells from spontaneous apoptosis. In contrast, the protection conferred by dextran sulfate was important in young mice, but it progressively vanished with age and disappeared when mice reached 16 months of age. Meanwhile, the susceptibility to dbcAMP- and CHX-induced apoptosis greatly augmented as mice grew older.
4. Discussion The present study demonstrates a direct effect of aging on the response of B lymphocytes to various stimuli affecting apoptosis and activation. Alteration of the antibody response in old individuals has been well established (Thorbecke et al., 1994; Miller and Kelsoe, 1995; Zhao et al., 1995) but was generally considered as the consequence of multiple interactions in the immune system (Proust et al., 1996; Yang et al., 1996; Burns and Goodwin, 1997). However, intrinsic defects of isolated cells in the B-cell lineage have also been demonstrated (Borghesi and Nicoletti, 1996). The sensitivity to endotoxin appears to increase with aging (Tschepen et al., 1994; Chorinchath et al., 1996; Tateda et al., 1996). With respect to the B-cell proliferative response, early reports have shown a decrease in the proliferation of whole spleen cells in response to LPS (Abraham et al., 1977; Averill and Wolf, 1985). The
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decline with age in mitogenic activity was first attributed to a reduction in the number of responding cells and not to an alteration in their capacity to divide (Abraham et al., 1977). In contrast, other data have suggested that this decline was primarily due to an intrinsic defect rather than to a decrease in B cell number (Averill and Wolf, 1985). We too observed a reduced proliferative response of unseparated spleen cells to LPS. In contrast, we found similar proliferation rate in purified B cells from both young and old mice, indicating that the proliferative capacity of B lymphocytes was not impaired with increasing age. This suggests that the age-related reduction in the proliferative response of whole spleen cells to LPS was mediated by the other cells present in spleen cells. Thus, macrophages have been reported to contribute to the reduced proliferative response of old splenic T cells to PHA through an increased production of prostaglandins (Beharka et al., 1997). In addition, upon stimulation with LPS, monocytes from elderly humans were reported to produce less IL-1 than monocytes from young subjects (McLachlan et al., 1995). Therefore, macrophages could exert a negative effect on the response of spleen cells to LPS through an increased production of prostaglandins or a reduced secretion of IL-1. Another alternative involves T cells which have been shown to contribute to the age-associated changes in the antibody repertoire of B cells (Song et al., 1997) and are known to provide less efficient help and more suppressive effects with aging (Engwerda et al., 1996; Miller et al., 1997; Segre and Segre, 1997). It has been reported that glucocorticoids exert antiproliferative activity on B lymphocytes (Sabbele et al., 1987); consistent with these findings we observed that dexamethasone greatly inhibited LPS-induced proliferation and, interestingly, inhibition was similar in old and young mice. It has also been found that the proliferative responses of Peyer’s patch B cells from aged and young mice to LPS were similar (Kawanishi and Joseph, 1992). Recent studies have reported that stimulation by LPS of the capacity of stable clonal B-cell populations to generate an Ig heavy chain diverse repertoire did not change with age (LeMaoult et al., 1997). These results argue for an age-independent responsiveness of B cells to LPS. However, the effect of LPS on expression of ALP decreased with age. This attenuated ALP expression appeared to be linked to an intrinsic age-associated defect of B cells to become activated since it was also observed in lymphocytes from LPS-hyporesponsive C3H/HeJ mice stimulated with other differentiation stimuli. Thus, dextran sulfate stimulation of ALP expression decreased with aging but IL-5, which was devoid of any activity on ALP induction by itself, synergised with dextran sulfate for increased ALP expression in both aged and young mice. ALP is considered as a marker of B-cell differentiation since its expression has been associated with the differentiation of B cells into antibody-producing cells (Burg and Feldbush, 1989; Marquez et al., 1990; Souvannavong et al., 1992, 1994). Furthermore, activation of cell surface ALP by specific antibody has been reported as an effective signaling for antibody production (Marty and Feldbush, 1993). Thus, our results suggest that the intrinsic defect of aged B cells affects the signaling pathway involved in cell differentiation rather than in B cell proliferation.
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Increasing evidence suggests that senescence could be associated with perturbations in the apoptotic process (Chrest et al., 1993; Ucker et al., 1994; Proust et al., 1996; Fulop et al., 1997; Warner, 1997). We observed that B cells obtained from untreated mice rapidly underwent apoptosis and that, unexpectedly, the rate of apoptosis was higher in young mice than in older animals. Contrasting results reported that spontaneous apoptosis of splenic B cells increased with age in normal mice but not in lpr and gld mice suggesting an involvement of fas in that phenomenon (Reap et al., 1995). The basis for discrepancy is unclear but may relate to differences in the experimental conditions; the authors used unseparated spleen cells and apoptosis was determined after an overnight culture by a biparametric cytometric analysis of DNA content in IgM + cells. We used purified B cells, thus avoiding the possibility of an environmental influence of other splenocytes. Although B cells from old mice presented lower rates of spontaneous apoptosis they appeared to be more susceptible than those from young mice to agents, such as dexamethasone, cycloheximide or dibuyryl cAMP, which are known inducers of apoptosis. In contrast, while the protective activity of LPS or IL-4 was not reduced with age, only B cells from young mice could be protected by dextran sulfate, a protection that could not be improved by IL-5 which did not appear to affect B-cell apoptosis. The present study demonstrates an intrinsic defect of B lymphocytes during aging leading to a modulation of their response to various stimuli: while their capacity to proliferate was maintained, their capacity to become activated or to undergo apoptosis was modified.
Acknowledgements We thank N. Esquirol for her excellent technical assistance and J.M. Bureau for his precious help in cytometric experiments. This work was supported by the Centre National de la Recherche Scientifique (CNRS-ERS 571) and the Association pour la Recherche sur le Cancer.
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Age-associated modulation of apoptosis and activation in murine B lymphocytes Vongthip Souvannavong a,*, Christophe Lemaire a, Karine Andre´au a, Spencer Brown b, Arlette Adam a a
CNRS-ERS 571, Institut de Biochimie, Baˆt. 430, Uni6ersite´ Paris-Sud, Orsay Cedex 91405, France b CNRS-UPR 40, ISV, Gif sur Y6ette, 91198, France Received 7 February 1998; received in revised form 8 April 1998; accepted 15 April 1998
Abstract We have investigated the influence of age on B-cell responsiveness. The present study showed that the B-cell mitogen, lipopolysaccharide (LPS), similarly stimulated the proliferation of purified B lymphocytes obtained from either young mice (3 months) or old mice (24 months). In contrast, expression of the differentiation marker, alkaline phosphatase (ALP), was about fourfold higher in young mice than in older mice upon stimulation with LPS or with dextran sulfate (DXS) and interleukin-5 (IL-5). The occurrence of apoptosis during aging was then studied: unexpectedly, spontaneous cell death was double in B lymphocytes from young mice compared to older animals. Stimulation with DXS with or without IL-5 rescued B lymphocytes from cell death in young mice but protection decreased with aging, and no longer occurred in 24-month-old mice B cells. Meanwhile, the protective activity conferred by IL-4 was maintained at similar levels throughout aging. However, B cells from old mice were more responsive to apoptosis induction with cycloheximide, dibutyryl cAMP and dexamethasone. Together, the present results indicate an age-associated alteration in apoptosis and activation of B lymphocytes which could contribute to the age-related decline of the immune response. © 1998 Elsevier Science Ireland Ltd. All rights reserved.
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Keywords: Murine B lymphocytes; Aging; Proliferation; Alkaline phosphatase; Apoptosis
1. Introduction It is well recognised that aging is associated with a decline in immune function leading to various infectious and neoplastic diseases in man as well as in experimental animals (Miller, 1991; Chiarugi et al., 1994; Pawelec et al., 1995; Rubin et al., 1996). This age-related deterioration has been attributed to many factors including environmental changes and a failure of senescent cells themselves (Thoman and Weigle, 1989; Hodes, 1995; Proust et al., 1996; Burns and Goodwin, 1997; Hodes, 1997; Warner, 1997). The age-related changes in the antibody response is well documented and has been associated with alterations in T-cell help, in accessory cells and also with intrinsic B-cell defects (Goidl et al., 1976; Burns et al., 1990; Miller and Kelsoe, 1995; Borghesi and Nicoletti, 1996; Miller, 1996; Hodes, 1997; Segre and Segre, 1997). A more limited number of studies has been performed to evaluate the influence of age on the proliferative response of B cells. A decrease of the proliferative response of unseparated spleen cells to B-cell and T-cell mitogens has been reported leading to conflicting interpretations on whether age-related reduction was really due to an intrinsic defect of B cells or to a decrease in the number of responding cells (Abraham et al., 1977; Averill and Wolf, 1985). The proliferative response of purified human peripheral blood B cells to stimulation with anti-IgM or anti-m, with the polyclonal activator SAC, or with mAb against CD20 or CD40, has been reported to be reduced in elderly subjects (Whisler et al., 1991). In contrast, an age-related increase of the proliferative response of B lymphocytes to stimulation with anti-CD40 antibody has been demonstrated in mice in spite of the expression of comparable levels of CD40 in young and in old animals (Song et al., 1997). Paradoxically, while overall immune responsiveness decreases with age, autoimmunity appears to increase (Goidl et al., 1981; Zhao et al., 1995). The age-related qualitative and quantitative changes in antibody production, reflecting alteration in B-cell repertoire and generally associated with deficiency in T-cell help (Song et al., 1997), might also result from a failure of autoreactive cells to undergo cell death (Baixeras et al., 1994; Maclennan, 1995). Apoptosis, considered as physiological cell death as opposed to degenerative cell death (Kerr et al., 1972), plays a fundamental role in the homeostasis of the immune system and is essential in the negative selection and deletion of autoreactive cells (Golstein et al., 1991; Cohen et al., 1992). Many studies have been performed on the T-cell lineage but there is limited information on the role of aging in B-lymphocyte susceptibility to apoptosis. Alkaline phosphatase (ALP, EC 3.1.3.1) belongs to the family of glycosyl-phosphatidylinositol (GPI)-anchored proteins which play an important role in signal
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transduction (Ferguson and Williams, 1988; Anderson, 1993). ALP expression has been associated with B-lymphocyte differentiation (Burg and Feldbush, 1989; Marquez et al., 1990; Souvannavong et al., 1992, 1994) and with the transduction of activation signals (Marty and Feldbush, 1993). ALP was used here as a marker of B-cell differentiation. In the present study, the influence of aging on B-cell apoptosis and activation has been investigated. The results indicated that the proliferative response of purified B lymphocytes was maintained through aging, while their apoptotic susceptibility and capacity for activation were modified.
2. Materials and methods
2.1. Animals Female BDF1 (C57BL/6 ×DBA2)F1 and C3H/HeJ mice were purchased from IFFA CREDO (L’Arbresle, France) and CSAL (La Source, France), respectively, at 8 weeks of age and maintained until use in a sterile microisolator cage system at our central animal care facility.
2.2. Antibodies, interleukins and reagents Recombinant mouse IL-4 and IL-5 were obtained from Genzyme (Cambridge, MA). Monoclonal anti-Thy-1.2, anti-L3T4 (GK1.5), anti-Lyt-2 and anti-Lyt-3 were obtained from Serotec (Oxford, UK). The culture medium used throughout was RPMI 1640 supplemented with 25 mM Hepes, 2 mM L-glutamine, standard antibiotics, 50 mM 2-mercaptoethanol and 8% heat-inactivated fetal calf serum (Gibco, Paisley, Scotland). Cycloheximide (CHX), 6-diamidino-2-phenylindol (DAPI), dexamethasone (DEXA), dextran sulfate (DXS, 500000 MW), dibutyryl cAMP (dbcAMP), dibutyryl cGMP, fluorescein-diacetate (FDA), and lipopolysaccharide (LPS, from Salmonella enteritidis) were purchased from Sigma (St. Louis, MO). RU 486 was a kind gift of Roussel Uclaf, Romainville, France.
2.3. Preparation of purified B cells and culture B lymphocytes were purified as previously described (Souvannavong et al., 1994). Briefly, splenocytes (1 – 2×107/ml in RPMI medium containing 8% FCS) were treated with a cocktail of anti-T-cell monoclonal antibodies at 4°C for 45 min followed by incubation with a 1:10 dilution of low-tox rabbit complement at 37°C for 50 min. Cells were layered on top of a discontinuous Percoll gradient and cells at the 55 – 65% interface were recovered. More than 98% of these cells were found to be surface Ig-positive with FITC-conjugated rabbit anti-mouse Ig antibodies; such purified B cells were unresponsive to Concanavalin A, but retained full responsiveness to LPS. B cells, at 5× 105 cells/ml, were cultured in 24-well plates (Nunclon) in 6% CO2 at 37°C, in medium alone or in the presence of stimuli. These
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Fig. 1. Effect of LPS on the proliferation of splenic and purified B cells. Spleen cells (SC) or purified B cells from old and young BDF1 mice were stimulated with LPS. (A) Kinetics of [3H]TdR incorporation by cells stimulated with LPS (5 mg/ml) for 1 – 4 days; (B) dose-effect of LPS at day 3. (C) Purified B cells from young (shaded bars) and old (solid bars) mice were cultured for 3 days in the presence of LPS (5 mg/ml) with or without dexamethasone (5 nM) and RU 486 (100 nM). Cells (1 ×105/well) were cultured in 96-well culture plates and pulse labeled with 18 kBq/well of [3H]TdR for the final 6 h. Results are expressed as mean cpm9 SD for pooled triplicate cultures from three experiments. Mean values were compared using the Student’s t-test (old versus young mice), differences being significant (*) for P= 0.05, and highly significant (**) for P= 0.001.
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cells were used for gel electrophoresis of DNA or for determination of ALP activity, and cells fixed in 70% ethanol were kept at − 20°C until analysis of DNA content by flow cytometry.
2.4. Cell proliferation Whole splenocytes or purified B cells (1× 105/0.2 ml) were cultured in 96-well plates in medium alone or in the presence of stimuli. Proliferation was assessed at the indicated days by pulsing cultures with 0.5 mCi of [3H]thymidine ([3H]TdR specific activity, 5 Ci/mmol, 1 mCi= 37 kBq, Amersham, Les Ulis, France) 6 h before harvesting. Results are expressed as the mean cpm9S.D. for triplicate cultures.
2.5. ALP assay Cellular alkaline phosphatase activity was determined after 5 days of culture by p-NPP hydrolysis. Briefly, 100 ml of p-NPP dissolved at 1 mg/ml in buffer consisting of 0.1 M diethanolamine, 1 mM MgCl2, and 0.5% Triton X-100, pH 10.5, was added to cell pellets corresponding to 5×104 cells. After a 20-min incubation at 37°C, the reaction was stopped by addition of 50 ml 0.3 N NaOH. Results are reported as the mean arbitrary absorbance (A) units for triplicate wells measured at 405 nm, using a Dynatech microplate reader (MR 700).
2.6. Apoptosis analysis Apoptosis was determined by three methods including a morphological study, a quantitative analysis of apoptotic hypodiploid cells by flow cytometry, and DNA electrophoresis. Cell viability was assessed by FDA staining. Apoptosis was determined morphologically after staining with DAPI. Morphological criteria for apoptotic cell death were chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. DNA fragmentation was assessed by electrophoresis as previously described (Sarih et al., 1993). Briefly, DNA was extracted from lysates of 5 × 105 B cells after treatment with proteinase K and RNAse A. Electrophoresis was performed in agarose gel containing 1 mg/ml ethidium bromide and DNA visualised in UV light. The rate of apoptosis was determined by flow cytometry (Darzynkiewicz et al., 1992). Ethanol-fixed cells (5× 105 cells/ml) were washed twice with HBSS and stained with DAPI (2 mg/ml) at 37°C for 30 min. DNA analysis was then performed on a PARTEC CA II flow cytometer (Chemunex, Maisons-Alfort, France) equipped with a 100-W mercury lamp. Fluorescence at 455 nm was recorded as a function of relative DNA content. Each cytogram was generated till the analysis reached at least 15000 cells. The percentage of apoptotic cells was determined from the sub-G1 peak on cytograms.
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Fig. 2. Effect of age on ALP expression by B lymphocytes. (A) Purified B cells from young (3 months) and old (24 months) normal mice were stimulated with LPS (0.01 – 25 mg/ml). (B) Purified B cells obtained from 2–24-month-old C3H/HeJ mice were cultured in the presence or absence of DXS or DXS+ IL-5 as indicated. ALP activity was determined after 5 days of culture in 5 × 104 cells by p-NPP hydrolysis and expressed as arbitrary absorbance units at 405 nm. Standard deviation bars are given.
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2.7. Statistical analysis Results are expressed as the arithmetic mean9 S.D. for pooled triplicate cultures from three experiments. Mean values were compared by using Student’s t-test for unpaired observations, and the level of significance is indicated in Figs. 1–5, a P-value of 0.05 being considered as significant, and of 0.001 as highly significant.
Fig. 3. Apoptosis of B lymphocytes in young and old mice. Purified B cells from young (3 months) and old (24 months) normal BDF1 mice were cultured for 20 h. (A) Cells were stained with DAPI and analyzed for DNA content; frequency of nuclei with hypodiploid DNA is proportional to the percentage of apoptosis and is used for quantitation. (B) Apoptosis, as demonstrated by laddering of DNA from young (lanes 1–3) and old (lanes 4–6) mice, of B cells cultured in the absence (lanes 1 and 4) or in the presence of CHX (2.5 mg/ml, lanes 2 and 5) or dbcAMP (100 mM, lanes 3 and 6).
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Fig. 4. Age-related effect of various stimuli on B-cell apoptosis. B cells from young (shaded bars) and old (solid bars) normal BDF1 mice (A) and LPS-hyporesponsive C3H/HeJ mice (B) were cultured for 20 h with or without LPS (2.5 mg/ml), dexamethasone (5 nM), DXS (5 mg/ml) and IL-5 (5 units/ml), IL-4 (20 units/ml), CHX (2.5 mg/ml), dbcAMP (100 mM), or dbcGMP (100 mM), as indicated. Quantification of apoptosis was performed by determination of sub-G1 peak (see Fig. 3A). Results are expressed as the percentage of controls. Statistical analysis was performed as described in Fig. 1.
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3. Results
3.1. LPS-induced proliferation is similar in B cells from young and old mice It has been reported that the proliferative response of whole spleen cells to T-cell and B-cell mitogens declined with age (Abraham et al., 1977; Averill and Wolf, 1985). We thus examined whether the response of purified B lymphocytes was similarly affected by aging. Fig. 1A shows that the kinetics of the proliferative response of both spleen cells and B cells to LPS were similar in old and in young mice, with an optimal response between day 2 and day 3. Furthermore Fig. 1B shows that, in a dose-dependent way, LPS increased the proliferation of splenocytes, cell proliferation at the effective dose (1 mg/ml and above) being in young mice (3 months) about double that in old mice (24 months). We then compared the response of purified B cells from young and old mice. The dose-response curve showed that proliferation rates were similar in B cells obtained from either young or old mice. In addition, LPS-induced proliferation was inhibited to a similar extent by the synthetic glucocorticoid, dexamethasone, in young and in old mice. This activity was mediated by glucocorticoid receptors since it was totally inhibited by the antagonist RU 486 (Fig. 1C).
Fig. 5. Influence of various stimuli on apoptosis of B cells from mice of varying ages. B cells were obtained from C3H/HeJ mice of increasing ages (3 – 24 months) and cultured for 20 h with or without DXS, IL-5, IL-4, CHX or dbcAMP. Quantification of apoptosis was determined as described in Fig. 3 and expressed as the percentage of controls. Standard deviation bars are given.
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3.2. ALP production by acti6ated B cells declines with increasing age Expression of ALP is considered as a marker of the differentiation of B lymphocytes. In the present study, expression rate of ALP was determined in LPS-stimulated B cells from both young (3 months) and old (24 months) normal BDF1 mice. Fig. 2 shows that LPS (10 ng–25 mg/ml), dose-dependently increased ALP activity in B cells from young mice, a significant ALP activity being obtained with a dose as low as 100 ng/ml and optimal activity being reached at 2.5 mg/ml LPS and maintained for higher doses. LPS was much less effective at stimulating ALP activity in B cells from old mice, with only a marginal increase whatever the dose used (Fig. 2A). To investigate further this considerable age-associated reduction of ALP expression, we analysed the response of LPS-hyporesponsive C3H/HeJ mice of increasing ages (2 – 24 months). B lymphocytes were treated with the polyclonal B-cell activator, dextran sulfate, and/or IL-5 which have been previously found to stimulate optimal ALP activity (Souvannavong et al., 1992). Results presented in Fig. 2B show that dextran sulfate, in an age-dependent way, stimulated the production of ALP by B cells. Its activity was further potentiated by IL-5 which by itself was unable to trigger ALP expression (data not shown). The capacity of B cells to produce ALP was age-dependent: it was maximal in young adults between 2 and 5 months of age and sharply decreased at 10 months and thereafter. ALP levels in stimulated B cells from old mice were low when compared to those obtained from B cells of young mice, nevertheless, they were higher than background levels of ALP in non-stimulated control cells at any age.
3.3. Age-related changes in spontaneous and drug-modified apoptosis B lymphocytes from young adults (3 months) and from old mice (24 months) were cultured with or without various stimuli known to affect apoptosis. Experiments were performed in parallel in normal BDF1 mice and in LPS-hyporesponsive C3H/HeJ mice. When similar results were obtained for both strains, only those obtained from one strain are presented. The viability of B cells derived from young and old mice was greater than 98% at the initiation of cultures. Apoptosis was evaluated by morphological observation after DNA staining with DAPI (not shown), by flow cytometric analysis of sub-G1 DNA content and by DNA gel electrophoresis. As shown in Fig. 3A, apoptosis spontaneously occurred after 20 h of incubation: cell death appeared to be greater in B cells from young mice (about 35%) than in older mice (about 20%). Apoptosis was accompanied by DNA fragmentation (Fig. 3B), this being higher in young (lane 1) than in old (lane 4) mice. Consistent with previous findings (Illera et al., 1993; Newell et al., 1993), we observed that the translation inhibitor, cycloheximide, and dibutyryl cAMP stimulated apoptosis; moreover, cell death of old mice appeared to be the most affected in spite of lower levels of spontaneous apoptosis (lanes 5 and 6 versus lanes 2 and 3).
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We then investigated whether protection against apoptosis was also affected by aging (Fig. 4). In agreement with previous reports on the repression of apoptosis in B cells (Norvell et al., 1995; Ashman et al., 1996), we observed that dextran sulfate and the cytokine, IL-4, reduced apoptosis of B cells in both mouse strains. As expected, LPS did not affect apoptosis in C3H/HeJ mice (Fig. 4B) but it protected normal B cells in an age-independent way (Fig. 4A). Stimulation with DXS and IL-5 protected young B cells from apoptosis (50 and 30% of controls for BDF1 and C3H/HeJ cells, respectively). The protection by DXS and IL-5 was very low in old normal mice and not significant in old C3H/HeJ mice. In contrast, the protective effect of IL-4 was independent of age in both strains (60 and 50% of controls for C3H/HeJ and BDF1 mice, respectively). On the other hand, stimulation of apoptosis appeared to be higher in B cells of old mice than in those of young mice. Thus, dexamethasone and CHX induced up to fourfold apoptosis in old mice versus twofold in young mice. Similarly, B-cell apoptosis linked to cAMP increase was more notably enhanced in older than in younger mice. An increase of cGMP levels, however, only marginally affected apoptosis whatever the strain or the age of mice.
3.4. Influence of 6arious stimuli on apoptosis of B cells from mice of 6arying ages In order to better define the influence of aging on the susceptibility to apoptosis, we examined the response of B cells from mice of increasing ages (3, 5, 10, 16, and 24 months). B cells were cultured in the presence or absence of various stimuli, and apoptosis was determined by flow cytometry as previously described. The results presented in Fig. 5 show that, independently of age, IL-5 failed to affect apoptosis whereas IL-4 partially rescued B cells from spontaneous apoptosis. In contrast, the protection conferred by dextran sulfate was important in young mice, but it progressively vanished with age and disappeared when mice reached 16 months of age. Meanwhile, the susceptibility to dbcAMP- and CHX-induced apoptosis greatly augmented as mice grew older.
4. Discussion The present study demonstrates a direct effect of aging on the response of B lymphocytes to various stimuli affecting apoptosis and activation. Alteration of the antibody response in old individuals has been well established (Thorbecke et al., 1994; Miller and Kelsoe, 1995; Zhao et al., 1995) but was generally considered as the consequence of multiple interactions in the immune system (Proust et al., 1996; Yang et al., 1996; Burns and Goodwin, 1997). However, intrinsic defects of isolated cells in the B-cell lineage have also been demonstrated (Borghesi and Nicoletti, 1996). The sensitivity to endotoxin appears to increase with aging (Tschepen et al., 1994; Chorinchath et al., 1996; Tateda et al., 1996). With respect to the B-cell proliferative response, early reports have shown a decrease in the proliferation of whole spleen cells in response to LPS (Abraham et al., 1977; Averill and Wolf, 1985). The
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decline with age in mitogenic activity was first attributed to a reduction in the number of responding cells and not to an alteration in their capacity to divide (Abraham et al., 1977). In contrast, other data have suggested that this decline was primarily due to an intrinsic defect rather than to a decrease in B cell number (Averill and Wolf, 1985). We too observed a reduced proliferative response of unseparated spleen cells to LPS. In contrast, we found similar proliferation rate in purified B cells from both young and old mice, indicating that the proliferative capacity of B lymphocytes was not impaired with increasing age. This suggests that the age-related reduction in the proliferative response of whole spleen cells to LPS was mediated by the other cells present in spleen cells. Thus, macrophages have been reported to contribute to the reduced proliferative response of old splenic T cells to PHA through an increased production of prostaglandins (Beharka et al., 1997). In addition, upon stimulation with LPS, monocytes from elderly humans were reported to produce less IL-1 than monocytes from young subjects (McLachlan et al., 1995). Therefore, macrophages could exert a negative effect on the response of spleen cells to LPS through an increased production of prostaglandins or a reduced secretion of IL-1. Another alternative involves T cells which have been shown to contribute to the age-associated changes in the antibody repertoire of B cells (Song et al., 1997) and are known to provide less efficient help and more suppressive effects with aging (Engwerda et al., 1996; Miller et al., 1997; Segre and Segre, 1997). It has been reported that glucocorticoids exert antiproliferative activity on B lymphocytes (Sabbele et al., 1987); consistent with these findings we observed that dexamethasone greatly inhibited LPS-induced proliferation and, interestingly, inhibition was similar in old and young mice. It has also been found that the proliferative responses of Peyer’s patch B cells from aged and young mice to LPS were similar (Kawanishi and Joseph, 1992). Recent studies have reported that stimulation by LPS of the capacity of stable clonal B-cell populations to generate an Ig heavy chain diverse repertoire did not change with age (LeMaoult et al., 1997). These results argue for an age-independent responsiveness of B cells to LPS. However, the effect of LPS on expression of ALP decreased with age. This attenuated ALP expression appeared to be linked to an intrinsic age-associated defect of B cells to become activated since it was also observed in lymphocytes from LPS-hyporesponsive C3H/HeJ mice stimulated with other differentiation stimuli. Thus, dextran sulfate stimulation of ALP expression decreased with aging but IL-5, which was devoid of any activity on ALP induction by itself, synergised with dextran sulfate for increased ALP expression in both aged and young mice. ALP is considered as a marker of B-cell differentiation since its expression has been associated with the differentiation of B cells into antibody-producing cells (Burg and Feldbush, 1989; Marquez et al., 1990; Souvannavong et al., 1992, 1994). Furthermore, activation of cell surface ALP by specific antibody has been reported as an effective signaling for antibody production (Marty and Feldbush, 1993). Thus, our results suggest that the intrinsic defect of aged B cells affects the signaling pathway involved in cell differentiation rather than in B cell proliferation.
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Increasing evidence suggests that senescence could be associated with perturbations in the apoptotic process (Chrest et al., 1993; Ucker et al., 1994; Proust et al., 1996; Fulop et al., 1997; Warner, 1997). We observed that B cells obtained from untreated mice rapidly underwent apoptosis and that, unexpectedly, the rate of apoptosis was higher in young mice than in older animals. Contrasting results reported that spontaneous apoptosis of splenic B cells increased with age in normal mice but not in lpr and gld mice suggesting an involvement of fas in that phenomenon (Reap et al., 1995). The basis for discrepancy is unclear but may relate to differences in the experimental conditions; the authors used unseparated spleen cells and apoptosis was determined after an overnight culture by a biparametric cytometric analysis of DNA content in IgM + cells. We used purified B cells, thus avoiding the possibility of an environmental influence of other splenocytes. Although B cells from old mice presented lower rates of spontaneous apoptosis they appeared to be more susceptible than those from young mice to agents, such as dexamethasone, cycloheximide or dibuyryl cAMP, which are known inducers of apoptosis. In contrast, while the protective activity of LPS or IL-4 was not reduced with age, only B cells from young mice could be protected by dextran sulfate, a protection that could not be improved by IL-5 which did not appear to affect B-cell apoptosis. The present study demonstrates an intrinsic defect of B lymphocytes during aging leading to a modulation of their response to various stimuli: while their capacity to proliferate was maintained, their capacity to become activated or to undergo apoptosis was modified.
Acknowledgements We thank N. Esquirol for her excellent technical assistance and J.M. Bureau for his precious help in cytometric experiments. This work was supported by the Centre National de la Recherche Scientifique (CNRS-ERS 571) and the Association pour la Recherche sur le Cancer.
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