Mechanisms of Ageing and Development 102 (1998) 263 – 277 Relation between exploratory activity and immune function in aged mice: a preliminary study Monica De la Fuente a,*, Marta Minano a, Victor Manuel Victor a, ´ ˜ ´ ´ Monica Del Rio a, Maria Dolores Ferrandez a, Araceli Dıez b, ´ b Jaime Miquel a Department of Animal Physiology, Faculty of Biological Sciences, Complutense Uni6ersity, E-28040 Madrid, Spain b Department of Pharmacology, Uni6ersity of Alicante, Alicante, Spain Received 19 June 1997; received in revised form 23 October 1997; accepted 27 October 1997 Abstract Previous studies show that fast exploration of a T-shaped maze by mature mice may predict an above average longevity. Since the nervous and the immune systems work in a coordinated fashion, and it seems that these two homeostatic systems both influence organismic aging and suffer a senescent decline, we have performed a comparative study of the above behavioral parameter and different functions of three representative immune cells: lymphocytes, macrophages and natural killer (NK) cells obtained from old (76 9 1 weeks of age) female OF1-Swiss mice. At 70 weeks of age the mice were divided into a ‘fast’ and a ‘slow’ group, containing 100 and 0%, respectively, of animals able to explore the 50 cm-long first arm of the maze in 20 s or less. At 76 9 1 weeks of age the animals were sacrificed, the peritoneal cell suspensions were obtained and the immune organs (axillary nodes, spleen and thymus) were isolated. The following leukocyte functions were studied in peritoneal macrophages: adherence to substrate, mobility (spontaneous and chemotaxis), ingestion of particles and superoxide anion production whereas mobility, lymphoproliferative response to the mitogen Con A and NK activity were studied in the immune-organ leukocyte suspen- * Corresponding author. Tel.: @eucmax.sim.ucm.es +34 1 3944989 fax: + 34 1 3944935; e-mail: mondelaf 0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00015-3 264 M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 sions. The results show that the aged fast mice have better immune functions than the aged slow mice. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Immune function; Leukocytes; Exploratory behavior; Mice 1. Introduction An increasing amount of data supports the concept that the nervous and immune systems are closely interconnected and they mutually influence their respective functions through common mediators and receptors (Ader et al., 1990; Blalock, 1994; Madden and Felten, 1995). Furthermore, it seems that these two regulatory systems suffer an intrinsic senescent decline (Solana et al., 1991; Ortega et al., 1993; Ferrandiz et al., 1994; Pawelec, 1995; Pawelec et al., 1995; Saransaari and Oka, 1995; Zhang et al., 1995) that may play a central role in most age-related dysfunctions (Fabris, 1986, 1991). In aging humans and animals there is a relation between performance in certain behavioral tests and expected life span. Thus, in the longitudinal human aging study of the Baltimore Gerontotogical Center (Borkan and Norris, 1976) male subjects showing low scores (as compared to men of the same age) in a ‘tapping’ test (that measured reaction time) tended to die earlier than those showing a faster performance. On the other hand, rat and mouse studies, more closely related to the present study, show a correlation between behavioral reactions to novel environmental stimuli in an exploration test and longevity. The data indicate that the life span of the Brown Norway (BN) and Wistar Kyoto (WKY) strains is inversely related to the intensity of their response to a new environment. Moreover, the shorter-lived WYK rats exhibited higher basal activity of the peripheral sympathoadrenal catecholaminergic system in comparison to the longer-lived BN rats (Gilad and Gilad, 1995). In agreement with the above, unpublished studies from our laboratories suggest that interindividual differences in life span among members of Swiss outbred mouse populations may be related to their behavior in a simple T-maze test. Our data indicate that most mice which quickly explore the maze reach a longer life span than mice that take longer to accomplish this task. This agrees with the previous finding that animals which exhibit immobility or ‘freezing behavior’ (i.e. high levels of anxiety) when placed in a new environment usually show a short life span (Gilad and Gilad, 1995). In order to obtain additional information relevant to above, the present work offers a preliminary exploration of the relations between immune system function and T-maze performance. We have investigated three representative immune cells, i.e. lymphocytes, phagocytes and natural killer (NK) cells from two different populations of aged mice: those which explore the above maze in less than 20 s and those which take longer. M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 265 2. Materials and methods 2.1. Animals We have used female outbred Swiss (Iffa-Credo, France) mice (Mus musculus) which were 7 weeks old on arrival at our laboratory. The animals were randomly divided in groups of 10, and each group was housed in polyurethane boxes, maintained in cages containing 10 mice each, at a constant temperature (229 2°C) in sterile conditions inside an aseptic air negative-pressure environmental cabinet (Flufrance, Cachan, France) on a 12/12 h reversed light/dark cycle. All animals were fed water and standard Sander Mus (A.04 diet from Panlab L.S. Barcelona, Spain) pellets ad libitum. The diet was in accordance with the recommendations of the American Institute of Nutrition for laboratory animals. 2.2. Experimental groups At 11 months of age, the spontaneous exploratory behavior of each mouse was tested in a T-shaped maze (with arms 50 cm in length), in order to sort out the ‘fast’ mice (which completed the exploration of the first arm of the maze in 20 s or less) from the ‘slow’ mice (which required over 20 s). Then, 20 mice were regrouped in two groups. One group contained the ‘fast population’ and the other the ‘slow population’, with a fast/slow mouse ratio of 100/0 and 0/100, respectively. Every 2 weeks all animals were subjected to the T-maze test. At 17 months of age, 16 surviving mice (8 from each group) not showing pathological processes were sacrificed by cervical dislocation. 2.3. Collection of leukocytes The mice were killed by cervical dislocation according to the guidelines of the European Community Council Directives 86/6091 EEC, and peritoneal suspensions were obtained following a method previously described (De la Fuente, 1985). The abdomen was cleaned with 70% ethanol, the abdominal skin was carefully dissected without opening the peritoneum, and 4 ml of Hank’s medium (Sigma) adjusted to pH 7.4 were injected intraperitoneally. The abdomen was massaged and 90–95% of the injected volume was recovered. Flow cytometric analysis showed a proportion of 40% macrophages and 60% lymphocytes in the peritoneal suspension. The resting peritoneal macrophages were quantified and identified by their morphology and non-specific esterase staining. Both kinds of cells were counted and then adjusted to 5× 105 macrophages/ml or 5 × 105 lymphocytes/ml of Hank’s medium. Cellular viability, routinely determined before and after each experiment using the Trypan-blue exclusion test, was in all cases higher than 95%. For collection of immune organ leukocytes, after a peritoneal suspension was obtained from each animal, the axillary nodes, the spleen and the thymus were removed aseptically, freed of fat, minced with scissors and gently pressed through a mesh screen (Sigma). The cell suspension obtained from each organ was cen- 266 M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 trifuged in a gradient of Ficoll-Hypaque (Sigma) with a density of 1.070 (Boyun, 1968). Each halo of leukocytes was resuspended and washed three times in phosphate buffered saline (PBS) solution. Every cell suspension was separated in three aliquots for the mobility, proliferation and NK assays. The cell viability, measured by the Trypan blue exclusion test, was about 98%. 2.4. Assays of phagocytic function in peritoneal macrophages The assay of phagocytic function of the macrophages was carried out on the peritoneal suspensions. For the quantification of adherence capacity to the substrate, we observed the adherence to a smooth plastic surface because it resembles adherence to animal tissue. The method was carried out as previously described by us (De la Fuente et al., 1991). Briefly, aliquots of the peritoneal macrophage suspensions (adjusted to 105 cells/ml Hank’s medium) were placed in Eppendorf tubes and incubated 10, 20, 30 and 60 min at 37°C, and after gently shaking, the number of non-adhered macrophages was determined in Neubauer chambers. The adherence index, Al, was calculated according to the following equation: Al = 100-[(non-adherent cells/ml)/ (initial cells/ml)] × 100. The mobility assays (spontaneous mobility and directed mobility or chemotaxis) were determined according to a modification (De la Fuente et al., 1991) of the original technique described by Boyden (1962), which consists basically in the use of chambers with two compartments separated by a filter (Millipore) with a pore diameter of 3 vm. Aliquots of 0.3 ml of the peritoneal suspension (5× 105 cells/ml) were deposited in the upper compartment of the Boyden chambers. F-met-leu-phe (Sigma) (a positive chemotactic peptide in vitro), at 10 − 8 M, was placed in the lower compartment in order to determine chemotaxis. For spontaneous mobility, a Hank’s medium free of chemotactic factor was used. The chambers were incubated for 3 h at 37°C and 5% CO2, and after this time the filters were fixed, stained and both the chemotaxis and mobility indexes were determined by counting in an optical microscope (immersion objective) the total number of macrophages in one third of the lower face of the filters. The latex phagocytosis assay was carried out following the method described by De la Fuente (1985). Aliquots of 200 vl of peritoneal suspension were incubated in migration inhibitory factor (MIF) plates (Sterilin, Teddington, Middlesex, UK) for 30 min. To the adherent monolayer, after being washed with PBS, 20 vl latex bead (1.09 vm diluted to 1% PBS, Sigma) were added. After 30 min of incubation, the plates were washed, fixed and stained and the number of particles ingested by 100 macrophages was counted. The superoxide anion production, the first response in the respiratory burst, was evaluated assessing the capacity of this anion, produced by macrophages, to reduce nitroblue tetrazolium (NBT). It was carried out following the method described by De la Fuente et al. (1991) slightly modified as follows: aliquots of 250 vl of peritoneal suspension were mixed with 250 vl of NBT (1 mg/ml in PBS, Sigma). Twenty microliters latex bead suspension were added to the stimulated samples and M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 267 20 vl of PBS to the non-stimulated samples. After 60 min of incubation, the reaction was stopped, the samples were centrifuged, and the absorbance of the supernatants determined at 525 nm in a spectrophotometer (extracellular measure of superoxide anion production). The intracellular reduced NBT was extracted with dioxan (Sigma) and, after centrifugation, the supernatant absorbance at 550 nm was determined (intracellular measure of superoxide anion production). 2.5. Assays of lymphocyte functions Adherence of lymphocytes was studied in peritoneal suspensions (adjusted at 5×105 lymphocytes/ml of Hank’s solution) following the same method indicated above for macrophages. Spontaneous mobility and chemotaxis were studied in peritoneal lymphocytes as well as lymphocytes obtained from axillary nodes, spleen and thymus, and in all samples adjusted to 5× 105 lymphocytes/ml of Hank’s solution. The assays were carried out as described above for macrophages. Lymphoproliferative response to mitogen was measured in the aliquot of the leukocyte suspension resuspended in RPMI 1640 enriched with L-glutamine (Gibco Canada Ltd., Burlington, Ontario) and supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco) and gentamicin (100 vg/ml, Gibco), and adjusted to 106 cells/ml medium. We followed a standard method previously described (Del Rio et al., 1994) with slight modifications. Aliquots of 200 vl of the lymphocyte suspensions were dispensed in 96 well flat-bottomed microtiter plates (Costar, Cambridge, MA) and 20 vl of Con A (1 vg/ml) or 20 vl of Hank’s solution (controls) were added for the measurement of spontaneous or mitogen-induced lymphoproliferation, respectively. After 48 h of incubation at 37°C in an atmosphere of 5% CO2, 0.5 vCi/well [3H]thymidine was added to each well. After another 24 h incubation, lymphocytes were harvested in a semiautomatic microharvester and the thymidine uptake was measured in a i-counter (LKB, Uppsala, Sweden) for 1 min. Results were expressed as percentage of [3H]thymidine uptake (cpm), giving the 100% value to the cpm obtained in samples without mitogen. 2.6. NK acti6ity assay An enzymatic colorimetric assay was used for cytolysis measurements of target cells (Cytotox 96 TM Promega, Boehringer Ingelheim) based on the determination of lactate dehydrogenase (LDH) using a tetrazolium salt. This technique has been shown to provide identical values (within experimental error) to those obtained by parallel 51Cr release assays by our group and by other authors (Decker and Lohmann-Matthes, 1988; Del Rio and De la Fuente, 1995). Murine YAC-1 cells were used as target cells. These cells were maintained in complete medium (RPMI 1640 plus 10% FCS, Gibco) at 37°C, 5% CO2 and saturated humidity, being checked and counted periodically. Target cells were seeded in 96-well U-bottom culture plates (Costar) at 104 cells/well in 1640 RPMI without phenol red. Effector cells (leukocytes from axillary nodes, spleen and thymus) were added at 105 268 M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 cells/well. The effector/target rate used, 10/1, was found by us (Del Rio and De la Fuente, 1995) to be responsible for similar results to those obtained in previous work with radioactive techniques (De la Fuente et al., 1993). The plates were centrifuged at 250×g for 4 min to facilitate cell to cell contacts and then they were incubated for 4 h at 37°C. After the incubation, LDH activity was measured in 50 ml/well of the supernatants by addition of the enzyme substrate and absorbance was recorded at 490 nm. Four kinds of control measurements were performed: a target spontaneous release, a target maximum release, an effector spontaneous release and a volume correction control, in order to adjust the volume change caused by the addition of lysis solution to maximum release control wells. To determine the percentage of target cells killed, the following equation was used: % lysis= ((E − ES) − TS/M− TS) × 100 where E= mean of absorbance in the presence of effector cells, ES =mean of absorbance of effector cells incubated alone, TS= mean of absorbance in target cells incubated with medium alone, and M =mean of maximum absorbance after incubating target cells with lysis solution. 2.7. Statistical analysis The data are expressed as the mean 9 S.D. of the values from the number of experiments shown in the figures. The data were evaluated statistically by the Student’s t-test, P B 0.05 being the minimum significant level. The normality of the samples was confirmed by the Kolmogorov–Smirnov test. 3. Results The percentages of slow and fast mice composing each group along remained about the same throughout the study (i.e. from the 11th to the 17th month of age). Moreover, because of the ‘training’ effect of the frequent testing, most fast mice became even faster over the course of the investigation. As regards the macrophage functions (Figs. 1–3), they seemed better in the fast than in the slow mice. Thus, the adherence indexes (Fig. 1) of macrophages from fast mice were higher than those from slow mice, with the differences being statistically significant at 10 (P B 0.01), 20 and 30 (PB 0.05) min of incubation. Both the spontaneous mobility and the chemotaxis index (Fig. 2) were superior in cells from fast mice, with statistically significant differences (PB0.01) between fast and slow mice. By contrast, the two mouse populations did not differ in their phagocytosis index, which was 313957 for the fast and 3059 39 for the slow animals. Superoxide anion production (Fig. 3) showed higher intracellular values in the macrophages from the fast mice, with significant differences (PB 0.05) between fast and slow animals in the stimulated samples. Conversely, the extracellular production of this free radical in both stimulated and non-stimulated samples was significantly decreased (P B 0.05) in the macrophages from fast mice. Lymphocyte functions were also higher in the fast mice, with significant differences between the adherence index of fast and slow animals at 10 (PB 0.01) and 20 M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 269 (PB0.05) min of incubation (Fig. 4). Furthermore, the indexes of spontaneous mobility and chemotaxis of lymphocytes from peritoneum, axillary nodes, spleen and thymus were higher in the fast than in the slow animals (Fig. 5). The statistical significance for the differences in lymphocyte spontaneous mobility between fast and slow mice was P B 0.05 for all above mentioned locations and PB 0.01 for the differences in chemotaxis of cells obtained from peritoneum, axillary nodes and spleen. By contrast, no differences were seen in the chemotaxis of thymus lymphocytes. The proliferative response to Con A (Fig. 6) was significantly (P B 0.05) increased in the lymphocytes from fast mice. NK activity (Fig. 7) showed higher values in the leukocytes from fast mice, although a statistically significant difference was only found in thymus (PB0.05). 4. Discussion According to our data, most parameters of immune function show lower values in the slow than in the fast mice. Furthermore, since it is well known that mouse Fig. 1. Adherence capacity of peritoneal macrophages from slow and fast female Swiss mice at 10, 20, 30 and 60 min of incubation. Each data represents the mean9S.D. of eight values (adherence indices) corresponding to eight animals, each value being the mean of duplicate assays. *P B 0.05; **PB0.01 with respect to values of slow mice. 270 M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 Fig. 2. Spontaneous mobility and chemotaxis of peritoneal macrophages from slow and fast female Swiss mice. Each column represents the mean9 S.D. of eight values (number of macrophages per filter) corresponding to eight animals, each value being the mean of duplicate assays. **PB 0.01 with respect to values of slow mice. aging is accompanied by a decreased exploratory drive (Ordy et al., 1964), the slow mice can be considered ‘biologically older’ than their fast counterparts. As regards the immune system, while most investigations of age-related changes in this system have focused mainly on lymphoproliferation that is decreased (Makinodan et al., 1969; Walford, 1969; Solana et al., 1991; Pawelec, 1995; Pawelec et al., 1997), whereas less attention has been paid to other immune functions such as lymphocyte adherence and mobility and NK activity and phagocytosis (Ortega et al., 1993; Sansoni et al., 1993; De la Fuente et al., 1995; Ferrandez and De la ´ Fuente, 1996). Nevertheless, the extant data justify the view that our aged fast mice have a better preserved immune system than the aged slow mice. A higher activity of NK cells, which lyse tumor cells and have important regulatory functions (Berke, 1989), is a favorable trait of fast animals. Moreover, the high indexes of adherence and mobility, the first and crucial functions involved in the immune and inflammatory responses (Doherty et al., 1987; Springer, 1990) that are shared by lymphocytes Fig. 3. (see right) Intracellular (upper figure) and extracellular (bottom figure) superoxide anion production in peritoneal macrophages from slow and fast female Swiss mice. Each column represents the mean9 S.D. of eight values (absorbance of nitroblue tetrazolium reduction) corresponding to eight animals, each value being the mean of duplicate assays. *PB 0.05 with respect to values of slow mice. M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 Fig. 3. 271 272 M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 Fig. 4. Adherence capacity of peritoneal lymphocytes from slow and fast female Swiss mice, and at 10, 20, 30 and 60 min of incubation. Each data represents the mean 9S.D. of eight values (adherence indices) corresponding to eight animals, each value being the mean of duplicate assays. *P B0.05; **PB0.01 with respect to values of slow mice. and phagocytes (Mackay and Imhof, 1993), found in fast animals, indicate that these animals have better phagocytic cells for the defense of the organism against pathogenic agents (Ortega et al., 1993) and a better migration capacity of lymphocytes searching for antigens. It is especially interesting that while the macrophages of fast mice produce higher levels of intracellular superoxide (which exerts a favorable effect on their phagocytic activity), the noxious inflammation causing release of superoxide is lower in these fast mice. Furthermore, a better lymphoproliferative response to mitogen in vitro represents a better antigen response in vivo, which is essential for an adequate immune response. A relation between early immunosenescence and short life span has been shown by previous work from other laboratories. The well-known contribution of immune decline to the impaired resistance to infectious and neoplastic diseases, both in experimental animals and in human subjects, supports the concept that an assessment of immune function can play a useful role in the prediction of morbidity and mortality of human populations (Pawelec et al., 1995). Life span does not show a correlation with any specific immune parameter (Lehtonen et al., 1990), but recent studies suggest that the analysis of a number of parameters may reveal correlations with the mortality rates of aged populations. Thus, it seems that the role of well M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 273 Fig. 5. Spontaneous mobility (upper figure) and chemotaxis (bottom figure) of lymphocytes from peritoneum, axillary nodes, spleen and thymus. Each column represents the mean 9S.D. of eight values (number of macrophages per filter) corresponding to eight animals, being each value the mean of duplicate assays. *PB 0.05; **PB 0.01 with respect to values of slow mice. 274 M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 preserved immune functions in the prevention of death becomes pre-eminent in subjects who have reached an advanced age (Pawelec et al., 1995). As regards our behavior data, since ‘freezing’ is linked to hyper-reactivity to environmental stressors (Gilad and Gilad, 1995), we can assume that the slow mice are more prone to sustain chronic stress throughout their lives because of social interaction with cagemates, handling by investigators and caretakers and other aspects of their environment. In agreement with previous research showing a correlation between stress, on one hand, and immunodepression (Ader et al., 1990) and aging (Ordy et al., 1964; Stein-Behrens and Sapolsky, 1992) on the other hand, both hyper-reactivity to a stressful stimulus and a less than optimal immune function may predict a short life span. The cellular and molecular processes that are responsible for the parallel age-related decline of the immune system and the central nervous system, and its effect on longevity are not well understood. The data reviewed by Felten et al. (1991) suggest that the hypothalamus, the limbic forebrain structures and the brain stem central autonomic nuclei play an essential role in the functional coordination of the immune system and the central nervous system. Moreover, noradrenergic sympathetic nerves, through direct innervation of immune organs, provide an anatomical Fig. 6. Proliferative response to the mitogen Con A of lymphocytes from axillary nodes, spleen and thymus. Each column represents the mean 9S.D. of eight values (percentage of [3H]thymidine uptake, cpm, giving the 100% value to the cpm obtained in samples without mitogen) corresponding to eight animals, each value being the mean of duplicate assays. *P B0.05 with respect to values of slow mice. M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 275 Fig. 7. NK activity of axillary nodes, spleen and thymus. Each column represents the mean 9S.D. of eight values (percentage of lysis) corresponding to eight animals, each value being the mean of duplicate assays. *P B0.05 with respect to values of slow mice. link between the nervous and the immune system (Felten and Felten, 1991). A role for declining noradrenergic innervation in immune senescence is suggested by the similarity between alterations of immune response with age and the effect of acute sympathetic denervation in young adults (Ackerman et al., 1991). Even more relevant to our present findings are the following comments by Ackerman et al. (1991): ‘The finding of parallel age-associated declines in sympathetic noradrenergic innervation of lymphoid organs and immune function suggests that alterations in the ability of the nervous system to signal the immune system through direct neural pathways may play a role in senescence of the immune system. No studies have tested this hypothesis directly; however, our studies of mice demonstrated a close correlation between the life span of the animal, onset and progression of changes in immune function and the decline of noradrenergic innervation of the spleen. The marked differences in timing of the spleen. The marked differences in timing of these events in different strains of mice support the notion than intrinsic genetic factors may control one or all of these processes’. It is probable that, in agreement with these views, the slow (hyperreactive) mice suffer an early dysfunction in the above mentioned nervous structures which in turn impairs immune competence and increases the rate of aging of both systems, with 276 M. De la Fuente et al. / Mechanisms of Ageing and De6elopment 102 (1998) 263–277 resulting shortening of life span. This hypothesis is in agreement with the finding of an abnormally low number of synaptic profiles in layer III of the frontal cortex in aged rats showing poor performance in a maze in comparison to good performers, both young and aged (Klein, 1983). We feel that further comparative studies on fast and slow mice may help to clarify the above issues, which are related to fundamental mechanisms of cellular and functional aging. 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