Peptides 21 (2000) 1403–1409 Age-related changes in the neuropeptide Y effects on murine lymphoproliferation and interleukin-2 production S. Medinaa, M. Del Rı́oa, A. Hernanzb, M. De la Fuentea,* a Departamento de Biologı́a Animal II (Fisiologı́a Animal), Facultad de Ciencias Biológicas, Universidad Complutense, Madrid, Spain b Servicio de Bioquı́mica, Hospital La Paz del Insalud, Madrid, Spain Received 17 February 2000; accepted 18 May 2000 Abstract Neuropeptide Y (NPY) modulates several aspects of the immune response but it is not known whether NPY responsiveness is altered with aging. In this work, the in vitro effect of NPY at concentrations ranging from 10214 M to 1027 M on lymphoproliferation has been studied in spleen, axillary node and thymus leukocytes from young, adult, mature and old BALB/c mice. The spontaneous proliferation of spleen lymphocytes from young mice was significantly stimulated by NPY. In response to the mitogen Con A, lymphoproliferation and IL-2 release by lymphocytes were inhibited significantly by NPY, these effects disappearing with aging. The results show that NPY is a modulator of lymphoproliferation and that this effect disappears progressively with age. Moreover, this regulatory role of NPY may be carried out through a decrease in IL-2 production. © 2000 Published by Elsevier Science Inc. Keywords: Aging; Neuropeptide Y; Lymphoproliferation; IL-2; Mice 1. Introduction Age-related changes in the immune system can result in an inappropriate response to antigens [24,33,37] leading to a greater susceptibility to diseases and death. The involution of the thymus gland is the most obvious age-related change in the immune system [32]. Accordingly, one of the most marker age-related alterations in the immune cells has been reported in the T lymphocytes, concretely in the lymphoproliferative response to mitogens, which is decreased in old subjects and animals [16,37]. Moreover, the low proliferative response of T cells is related to decreased interleukin 2 (IL-2) production, which is involved in the lymphoproliferative response to mitogens [16]. The immune system is in close intercommunication with the nervous system, and changes in the relationship between both systems have been regarded as responsible for systemic aging [11]. There are studies revealing the presence of sympathetic fibers in both primary and secondary lymphoid organs [40], and changes with aging in this noradrenergic innervation of lymphoid tissues. Moreover, anatomic and * Corresponding author. Tel.: 134-91-3944989; fax: 134-91-3944935. E-mail address: mondelaf@eucmax.sim.ucm.es (M. De la Fuente). 0196-9781/00/$ – see front matter © 2000 Published by Elsevier Science Inc. PII: S 0 1 9 6 - 9 7 8 1 ( 0 0 ) 0 0 2 8 4 - 9 biochemical data show sympathetic denervation in the splenic tissue during senescence [1]. Neuropeptide Y (NPY), a peptide from the pancreatic polypeptide (PP) family with an important role as neurotransmitter and neuromodulator in the nervous system, is released by noradrenergic neurons in addition to norepinephrine [15]. NPY is secreted from nerve endings in the peripheral and central nervous system as well as in all the lymphoid organs where T cells reside [40]. Immunochemical studies have revealed the presence of NPY in sympathetic neurons from lymphoid tissue with particularly high concentrations in spleen [34]. Recent data implicate NPY in the modulation of immune responses in vivo and in vitro. Thus, this neuropeptide decreases or increases immune functions such as antibody responses in rats [14], phagocytic process [7], cytotoxic activity [25], adhesion and migration [22,26], proliferation [2,19,27,36] and cytokine secretion [2,21]. The present study examines the changes occurring with age in both types of proliferation, i.e.; spontaneous and in response to the mitogen Con A, of lymphocytes from spleen, axillary nodes and thymus of young, adult, mature and old mice, and the possible immunomodulatory role of NPY on this lymphocyte function. Since IL-2 is an essential cytokine for the control of proliferation and differentiation of immune system cells, and specifically for the lympho- 1404 S. Medina et al. / Peptides 21 (2000) 1403–1409 proliferative response to antigens or mitogens [29], its release by mitogen-activated lymphocytes from adult and old mice as well as the possible modulation that could be produced by NPY on IL-2 release were also analyzed. 2. Materials and methods 2.1. Animals Female BALB/c mice were purchased from Iffa Credo (France) and were bred until the ages of 12 6 2 (young), 24 6 2 (adult), 50 6 2 (mature) and 72 6 2 (old) weeks. All animals were maintained at a constant temperature of 22 6 2°C in sterile conditions inside an aseptic air negativepressure environmental cabinet (Flufrance, Cachan, France) on a 12 h light/dark cycle and fed Sander Mus (Panlab, Barcelona, Spain) and water ad libitum. Although in previous studies we observed that the estrous cycle phase of the female mice has no effect on this experimental assay, all females used in the present study were at the beginning of dioestrous. Mice were checked periodically to verify their pathogen-free condition and the animals that presented signs of malignancy were eliminated from the study. 2.2. Reagents Neuropeptide Y (NPY), gastrin-releasing peptide (GRP), concanavalin A (Con A) and Ficoll-Hypaque were purchased from Sigma (St. Louis, MA). Neuropeptides and mitogen were dissolved in phosphate-buffered saline (PBS) solution. RPMI 1640 medium enriched with L-glutamine, fetal calf serum (FCS) and gentamicin were obtained from Gibco (Paisley, Scotland). Trypan blue was purchased from Merck (Darmstadt, Germany) and 5-bromo-29deoxy-uridine ELISA kit (BrdU) from Roche Diagnostics (Basilea, Switzerland). The immunoassay for detection of IL-2 was obtained from R&D Systems (Minneapolis, USA). 2.3. Collection of leukocytes Each animal was sacrificed by cervical dislocation according to the guidelines of the European Community Council Directives 86/6091 EEC. Spleen, axillary nodes and thymus were removed aseptically, freed of fat, minced with scissors and gently pressed through a mesh screen (Sigma). The cell suspensions obtained were centrifuged in a gradient of Ficoll-Hypaque with a density of 1.070 g/ml. The material in the interface was resuspended in complete medium (RPMI 1640 enriched with L-glutamine and supplemented with 10% heat-inactivated FCS and gentamicin, 100 mg/ml), washed and the cell viability was measured using the trypan blue exclusion test, showing a viability of about 98%. 2.4. Proliferation assay The proliferation of lymphocytes, spontaneous and induced by the mitogen Con A, was determined in 72 h cultures. Eight mice of each age group were employed in the experiments. The samples were tested individually and thus, aliquots of 200 ml of spleen, axillary node and thymus leukocyte suspensions, adjusted to 1 3 106 cells/ml of complete medium, were dispensed in 96-well flat-bottomed microtiter plates. Then, 20 ml of NPY at concentrations ranging from 10214 M to 1027 M, 20 ml of PBS (controls) or 20 ml of GRP (10210 M) as neuropeptide control [10], were added to the cells in the spontaneous proliferation assay. To evaluate the proliferative response to mitogen, 20 ml of Con A-mitogen (1 mg/ml) or Con A plus 20 ml of NPY or GRP at the different concentrations above indicated were used. Plates were incubated 48 h at 37°C in an atmosphere of 5% CO2. Then, to measure proliferation, a 5-bromo29deoxy-uridine (BrdU) ELISA labeling and detection commercial kit was used. The BrdU ELISA was performed according to the manufacturer’s instructions. Briefly, cells were pulsed with 20 ml of 110 mM BrdU solution during the last 24 h of culture or Con A-stimulation, and thus BrdU was incorporated into freshly synthesized DNA. Then, following fixation of cells, cellular DNA was partially digested by nuclease treatment. Next, a peroxidase labelled antibody to BrdU that binds to BrdU was added. As a final step, the peroxidase substrate was added yielding a colored reaction product as a result of peroxidase enzyme activity. The absorbance of the sample (measured at 405 nm, with a reference wavelength at 490 nm) is directly correlated to the level of BrdU incorporated into cellular DNA. It has been demonstrated that BrdU ELISA, the method described above, is the most sensitive in comparison to other proliferation assays used for the assessment of T lymphocyte growth [23]. 2.5. Interleukin 2 (IL-2) release in response to Con A and NPY IL-2 release was measured in supernatants of lymphocyte cultures from six adult and six old mice tested individually, which were collected at 48 h prior to pulsing with BrdU. This procedure allowed assessment of both proliferation and lymphokine release from the same wells. Briefly, the culture plates were centrifugated and the cell-free supernatants collected and frozen at -20°C until assay. The determination of IL-2 release in supernatants was performed using a commercial immunoassay kit with recombinant mouse IL-2 as standard, a minimum detectable dose of mouse IL-2 of 3 pg/ml and a quantification limit of 1000 pg/ml. S. Medina et al. / Peptides 21 (2000) 1403–1409 1405 Table 1 Spontaneous proliferative response of spleen lymphocytes from young, adult, mature and old BALB/c mice to NPY Control NPY (M) 10214 10213 10212 10211 10210 1029 1028 1027 GRP 10210 M YOUNG ADULT MATURE OLD 0.14 6 0.02●● 0.18 6 0.03 0.19 6 0.02 0.13 6 0.02●●● 0.16 6 0.01 0.17 6 0.02 0.15 6 0.02 0.18 6 0.02* 0.21 6 0.03** 0.19 6 0.02** 0.17 6 0.02* 0.15 6 0.02 0.19 6 0.03* 0.17 6 0.12 0.17 6 0.02 0.17 6 0.02 0.18 6 0.02 0.19 6 0.02 0.19 6 0.02 0.17 6 0.02 0.17 6 0.02 0.22 6 0.03** 0.20 6 0.02 0.21 6 0.03 0.20 6 0.02 0.20 6 0.02 0.21 6 0.02 0.21 6 0.03 0.21 6 0.02 0.20 6 0.03 0.27 6 0.03** 0.11 6 0.02 0.14 6 0.03 0.14 6 0.03 0.12 6 0.02 0.14 6 0.03 0.14 6 0.02 0.14 6 0.02 0.14 6 0.02 0.17 6 0.02* Each value is the mean 6 SD of eight experiments performed in triplicate, corresponding each experiment to one animal. GRP at 10210 M was used as neuropeptide control. * p , 0.05 and ** p , 0.01 with respect to the controls of the corresponding age. ●● p , 0.01, ●●● p , 0.001 compared to adult control value. 2.6. Statistical analysis All values are expressed as the mean 6 SD of eight experiments corresponding to eight animals, each value being the mean of duplicate or triplicate assays. The data were statistically evaluated by the analysis of variance (ANOVA) for repeated measures and the Scheffe F-test for comparison between each two groups. 3. Results The spontaneous proliferative response values of spleen lymphocytes incubated in the presence of NPY at concentrations ranging from 10214 M to 1027 M are shown in Table 1. NPY significantly stimulated lymphocyte spontaneous proliferation in vitro at concentrations from 10211 M to 1028 M in young animals, with the highest effect being observed at 10210 M and 1029 M (P , 0.01). No significant differences were shown in adult, mature and old ages with respect to their corresponding control values. When lymphocytes were incubated with GRP (10210 M), as a positive peptide control previously described for this immune function [10,27], the spontaneous proliferation of lymphocytes from young and old (P , 0.05), as well as from adult and mature animals (P , 0.01), was stimulated. With respect to the effect of NPY on axillary node and thymus lymphocytes, no changes were observed in the spontaneous proliferation values as compared to their respective controls (0.13 6 0.02, 0.12 6 0.03, 0.14 6 0.02, 0.12 6 0.02 for axillary nodes, and 0.15 6 0.03, 0.13 6 0.02, 0.14 6 0.02, 0.12 6 0.02 for thymus in young, adult, mature and old mice, respectively). By contrast, GRP exerted a stimulatory effect on spontaneous proliferation in axillary nodes from adult (0.15 6 0.01, P , 0.001) and mature (0.16 6 0.01, P , 0.01) mice, and in thymus from adult (0.15 6 0.01, P , 0.01), mature (0.17 6 0.02, P , 0.05) and old (0.14 6 0.01, P , 0.05) animals. Comparing the control values on spontaneous proliferation of spleen lymphocytes at the different ages, we observed the highest absorbance values in adult and mature mice (Table 1). With respect to the spontaneous proliferation of axillary nodes and thymus (data given above), no age-related significant differences were found. The results of the effect of NPY on the proliferative response induced by Con A, a T-cell mitogen, at the optimal concentration of 1 mg/ml are represented for spleen, axillary nodes and thymus lymphocytes in Fig. 1. NPY decreased the Con A-induced proliferation of lymphocytes from spleen in young, adult and mature ages compared to Con A alone. In young mice, the effective concentrations ranged from 10210 M to 1028 M (P , 0.05). In adult mice, a larger range of NPY concentrations than in young mice resulted inhibitory for this function, with P , 0.01 from 10213 M to 1028 M and P , 0.05 for 1027 M. With respect to mature mice, the 10211 M and 10210 M (P , 0.01) and the 1029 M (P , 0.05) concentrations produced a significant decrease of the mitogen response. However, no significant differences with NPY plus Con A were found with respect to Con A alone in old age. Regarding the effect exerted by GRP, which is a negative control [10,27] for the lymphoproliferative response to Con A, the proliferation in adult and mature ages were significantly inhibited, with the absorbance values being 0.72 6 0.09 (P , 0.01) and 0.53 6 0.06 (P , 0.01), respectively. Regarding the proliferation of axillary node lymphocytes, NPY caused a decrease of lymphoproliferation in response to Con A in young animals at the 1029 M concentration (P , 0.05), while in adult and mature mice, the 10210 M (P , 0.01) and 1029 M (P , 0.05) concentrations produced a decrease in the proliferation absorbance values with respect to Con A. No significant differences with NPY as compared to Con A alone were observed in old age. In addition, the peptide used as control, GRP, decreased the Con A-induced proliferation percentage in adult (0.57 6 0.06; P , 0.001) and mature mice (0.49 6 0.04; P , 0.05). With respect to thymus, NPY decreased the 1406 S. Medina et al. / Peptides 21 (2000) 1403–1409 Fig. 2. Proliferative response absorbance values in response to Con A (1mg/ml) of lymphocytes from spleen, axillary nodes and thymus of young, adult, mature and old BALB/c mice. Each value is the mean 6 S.D. of eight experiments performed in duplicate, corresponding each experiment to one animal. ** P , 0.01, ***P , 0.001 with respect to the absorbance value obtained in adult mice. The absorbance values of thymus were highly significant (P , 0.001) with respect to the other locations. Fig. 1. Effect of NPY at the indicated concentrations on the proliferative response of spleen, axillary node and thymus lymphocytes from young, adult, mature and old BALB/c mice to Con A as mitogen (1mg/ml). Each value is the mean 6 S.D. of eight experiments performed in duplicate, corresponding each experiment to one animal. * P , 0.05, ** P , 0.01, *** P , 0.001 with respect to the Con A control of the corresponding age. proliferation in response to Con A in young (from 10212 M to 1028 M) and adult mice (from 10213 M to 1028 M), 10211 M being the concentration with the highest effect at both ages (P , 0.001). No significant differences with respect to Con A alone were observed when NPY was added to lymphocytes from mature and old ages. Moreover, GRP decreased the Con A-induced proliferation in adult and mature ages, 0.30 6 0.04 (P , 0.001) and 0.19 6 0.02 (P , 0.05), respectively. Fig. 2 represents the proliferation values in response to Con A, corresponding to the different ages and immune organs studied. It can be observed that lymphocyte proliferation in response to Con A decreased with aging in all the locations studied, with the young and adult ages showing the highest absorbance values of proliferation, whereas the mature and old ages showing the lowest. Moreover, thymus showed significantly (P , 0.001) lower proliferation values with respect to the other locations studied. Finally, since IL-2 production is required for proliferation and differentiation of activated T cells, we assessed the IL-2 release by lymphocytes obtained from spleen and axillary nodes of adult and old mice. In the supernatants obtained from spontaneous proliferation, no detectable levels of IL-2 were found in both ages and locations. Following incubation with Con A or Con A plus NPY (from 10212 M to 1027 M), the results (Fig. 3) showed that spleen and axillary node lymphocytes of adult mice incubated with mitogen and NPY released less IL-2 than those incubated with Con A alone. This diminution was statistically significant in a range of concentrations of NPY from 10212 M to 1027 M in spleen, with the highest effect at the 10210 M and 1029 M concentrations (P , 0.01). Regarding axillary nodes, there was a statistically significant decrease (P , 0.05) at the 10210 M and 1029 M concentrations. However, no differences in the release of IL-2 were observed in old mice for both locations, when the samples were incubated with Con A plus NPY with respect to those incubated with Con A alone. In addition, a comparison of the values from the different ages studied showed a significant decrease in the release of IL-2 with aging (P , 0.01) for both locations. 4. Discussion Lymphocytes are important regulator and effector cells and therefore their activation is essential for a majority of immune responses. During activation these cells undergo complex changes involving differentiation and proliferation. The polyclonal activation of T cells by the mitogen S. Medina et al. / Peptides 21 (2000) 1403–1409 Fig. 3. Effect of NPY on IL-2 release in response to Con A (1mg/ml) of spleen and axillary node lymphocytes from adult and old BALB/c mice. Each value is the mean 6 S.D. of six experiments performed in duplicate, corresponding each experiment to one animal. * P , 0.05, ** P , 0.01 with respect to the corresponding control value. ●● P , 0.01 with respect to the adult Con A control. Con A mimics their stimulation by antigens [35]. Moreover, the secretion of IL-2, the cytokine triggered primarily by this activation, promotes expansion of the T populations [29]. The senescence process is accompanied by aging of the immune system and it has been reported by many authors that T-lymphocyte proliferation shows a sharp drop [16,17] and that the synthesis of IL-2 declines [13] with age. Thus, the decrease in mitogen-induced proliferation in aged animals shown in the present work is in agreement with previous work [33,37]. The accumulation of memory T cells with respect to naive cells with age could be responsible for the fewer T cells which are activated in aged individuals in response to mitogens [31]. The thymus of mature and old mice was the location showing the smallest percentages of proliferation in response to mitogen, which is in agreement with the fact that the involution of the thymus gland is the most obvious age-related change in the immune system [32]. However, it has been hypothesized that the function of thymus may not be crucial in later life, or that it may be taken over by other organs [12]. This could explain that in 1407 the other immune locations studied in the present work the decrease in response to Con A was not as pronounced as in thymus. Different mediators of the nervous system appear to influence numerous immune functions, including natural killer activity, proliferation and migration of immune cells [2,25–27]. In addition neuropeptides have been shown to exert modulatory effect on antibody and cytokine production, and macrophage activation, although the effects observed are heterogeneous and often contradictory [9]. In general, sympathetic factors have been often reported to have an inhibitory influence on T lymphocyte functions [15]. Thus, somatostatin appears to inhibit proliferation of human T lymphocytes when stimulated by phytohemagglutinin (PHA), Con A or alloantigens [6]. Moreover, VIP [8] have been reported to inhibit lymphoproliferation in response to mitogens, whereas substance P appears to be the only neuropeptide with stimulatory effects [38]. Other peptides such as cholecystokinin (CCK) and GRP have been shown to stimulate the spontaneous lymphoproliferation, while they decrease the proliferative response to PHA and Con A in adult human and mouse lymphocytes [5,10,27]. In addition, NPY has been shown to inhibit the proliferation of adult rat lymphocytes in response to PHA [36], which is in agreement with the results obtained in the present study. In general, the most significant effects were found with the NPY concentrations from 10211 M to 1029 M. The fall in the response at the highest concentrations could indicate a process of cell desensitization that is characteristic of receptor “down-regulation” [30]. Regarding the possible changes with aging in the effects of neuropeptides on lymphocyte functions, changes in cytotoxic activity as well as in migration induced by CCK, GRP and NPY have been reported by our group [25,26]. Age-related changes in the communication between the nervous and immune systems have been reported in these articles, since the influence of neuropeptides on the function of immune cells from adult animals is lost with age. The proliferation in response to Con A was significantly decreased by NPY in young and adult ages in all the immune locations studied in the present work, as well as in mature age in spleen and axillary nodes. The diminution or absence of NPY effects in mature and old mice seems to confirm that there is a disturbed in the communication between the nervous and the immune system with aging. Since, in the present in vitro study a wide range of NPY concentrations show no effect in old animals, these results in addition to those published before suggest that there is a deficiency in the immune cells from older animals in their response to nervous signals. With respect to the effect of NPY on spontaneous lymphoproliferation, a slight enhancement was found in young spleen lymphocytes. Other work has reported that NPY is mitogenic for vascular smooth muscle cells at similar concentrations to those found to be effective in the present study [41]. However, other authors have shown no change 1408 S. Medina et al. / Peptides 21 (2000) 1403–1409 in the spontaneous lymphoproliferation induced by NPY [19,21]. The IL-2 action is of great importance for proliferative T-cell response [29] and it is well established that aging is related to an impaired production of IL-2 by in vitro stimulated T-cells in rodents and humans [28,39]. However, other authors found no differences in IL-2 production by splenic T cells from young mice compared with T cells from aged mice of different strains [20]. The IL-2 levels found in this study, which are in agreement with those reported in other work [3] in spleen cells from a different mouse strain, are lower in stimulated spleen and axillary node lymphocytes from old mice as compared to those from adult mice. This parallel decline in IL-2 release and lymphoproliferative response may be due to the lower number of Th1 cells found in aged animals [4]. With respect to the modulation by NPY of the IL-2 release, its decreased production is related to the modulatory action shown in the lymphoproliferative response to Con A. Other neuropeptides, such as CCK and gastrin, have also been shown to decrease PHA-induced IL-2 production and lymphoproliferation [5]. Moreover, a similar behavior has been observed with VIP [8]. In contrast, treatment with somatostatin, calcitonin gene-related peptide, substance P and NPY in the absence of any additional factor has been reported to induce a marked secretion of IL-2 [21]. In conclusion, the results of the present study suggest that NPY modulates in vitro the proliferative response of these cells to Con A as well as the production of IL-2. Since these T cell functions have a pivotal role in the immune response and they are the most clearly depressed with age [18], the modulation exerted on them by NPY may represent a significant action of this neuropeptide on the immune system. Thus, its lack of effect in old age could be explained by a dysfunction of lymphocytes to respond to nervous factors. It might be that the decrease of the lymphoproliferation caused by NPY represents a positive role of this neuropeptide since it stops proliferation when needed in adult mice. 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