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Changes with aging in the modulation of macrophages by norepinephrine
Mechanisms of Ageing and Development 118 (2000) 103 – 114 www.elsevier.com/locate/mechagedev
Changes with aging in the modulation of macrophages by norepinephrine E. Ortega a,*, J.J. Garcı´a a, M.C. Sa´ez a, M. De la Fuente b a
Department of Physiology, Faculty of Science, Uni6ersity of Extremadura, A6enida de El6a s/n, 06071 Badajoz, Spain b Department of Animal Biology (Animal Physiology), Facutly of Biological Sciences, Complutense Uni6ersity, 28040 Madrid, Spain Received 8 March 2000; received in revised form 28 May 2000; accepted 29 June 2000
Abstract The effect of aging on the norepinephrine (NE)-induced modulation of phagocytic and oxygen-dependent microbicidal processes of mouse peritoneal macrophages was studied. Phagocytosis of latex beads on culture plates and superoxide anion production was evaluated in young (12 weeks), adult (22 weeks), mature (48 weeks) and old (72 weeks) BALB/c mice after in vitro incubation with 10 − 12, 10 − 9, 10 − 7, 10 − 5 or 10 − 3 M concentrations of NE. The results indicate that the phagocytic response to NE is quite similar in young and mature mice, with increased phagocytosis after incubation with 10 − 7 and 10 − 3 M, and decreased phagocytosis with 10 − 5 M. Macrophages from adult mice increased their phagocytic capacity after incubation with the highest concentrations of NE (10 − 5 and 10 − 3 M) and macrophages from old animals only were stimulated with 10 − 3 M. In addition, it was found that usually NE increased the extracellular superoxide anion production in the absence of phagocytosis in adult mice. No statistically significant changes were found in intracellular superoxide anion levels, but an increase was seen after phagocytosis in macrophages from adult, mature and old animals, especially after incubation with 10 − 5 M. In conclusion, the results obtained indicate that the modulation of macrophages by NE does not only depend on the concentration of this neurotransmitter, but also on age. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Macrophages; Norepinephrine; Mice
* Corresponding author. Fax: +34-24-271304. E-mail address: [email protected] (E. Ortega). 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 6 0 - 3
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1. Introduction It is well-known that the activity of the immune system declines with age. Clinical evidence for immunosenescence is the high incidence of tumours in elderly people. Also significant is the greater susceptibility of the aged to infections from pathogens to which they had been relatively resistant early in life (Weigle, 1989). In fact, elderly subjects who keep their immune functions at an excepcionally high level, live longer and may even become centenarians (Hirokawa, 1998). In some cases, the impairment of the immune response with age is a result of defective T-cell and/or B-cell functions. The T-cell functions especially show a marked decline with age, which is related to the involution of the thymus (Hirokawa, 1998; Solana et al., 1991; Utsuyama et al., 1991; Kay, 1996). Moreover, macrophages must be also taken into account as part of the cellular collaboration established in the antigenic response. A dysfunction of these cells could also contribute to declining lymphocyte function in old age (Callard, 1978; Makinodan and Kay, 1980; Solana et al., 1991). Thus, macrophages from old mice secrete inadequate amounts of IL-1, leading to decreased stimulation of T-helper cells and thereby contribute to diminished IL-2 production (Solomon and Benton, 1994). However, some studies suggest that the macrophage function, unlike the lymphocyte function, does not decline with age (Makinodan and Kay, 1980; Sondell et al., 1990), and there has even been observed a stimulation of the capacity of the monocyte–macrophage system in old individuals (Heidrick, 1972; Finger et al., 1982; Kuroiwa et al., 1989; Ortega et al., 1993a), especially with respect to phagocytosis. A similar situation is also found in stress, which also stimulates the phagocytosis of macrophages; and, on the contrary, causes a decline in lymphocyte function. Indeed, phagocytes can be stimulated by stress hormones (Cupps and Fauci, 1982; Dantzer and Kelley, 1989; Dohms and Metz, 1991; Forner et al., 1994; Ortega et al., 1997). In addition, during old age individuals present an alteration of the regulatory systems, including a negative effect on neuroimmune –endocrine integration (Fabris, 1992). It has been reported that this dysregulation of immune homeostasis, leading to vulnerability and failure under stress, may be linked to changes in the cellular milieu in which neurons and immune and endocrine cells function (Solomon and Benton, 1994), and can contribute to immunosenescence. Macrophages show receptors for catecholamines, and it has been reported that macrophage function is modified by catecholamines and adrenergic agonists (Madden and Livnat, 1991). However while an immunossupresor effect of catecholamines on lymphocytes has been established (Madden and Livnat, 1991), an immmunomodulation of these sympatetic mediators for non-specific immune responses of macrophages is uncertain, and probably it would be related to the age of individuals. In view of the above, the purpose of this investigation was to study whether the response of macrophages to norepinephrine is modified by aging.
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2. Materials and methods
2.1. Animals We have used in our study young (12 weeks), adult (22 weeks), mature (48 weeks) and old (72 weeks) BALB/c mice (Mus musculus), maintained at a constant temperature (22°9 2°C) on a 12-h light/dark cycle and standard laboratory diet with free access to food and water. The mice were examined, and only those without signs of malignancy or other pathological manifestations were used in the experiments.
2.2. Collection of cells The abdomen was cleansed with 70% ethanol, the abdominal skin carefully dissected without opening the peritoneum, and 3 ml of Hank’s solution adjusted to pH 7.4 injected i.p. The abdomen was massaged and the peritoneal exudate cells were removed, with recovery of 90–95% of the injected volume of fluid. The cells (macrophages and lymphocytes) were counted and adjusted to a final concentration of 5× 105 macrophages per ml in Hank’s solution (Ortega et al., 1993b).
2.3. Effect of norepinephrine (NE) on phagocytic function of macrophages In order to study the effect of NE on the ingestion (phagocytosis) capacity for latex beads of peritoneal macrophages, 200 ml of macrophage suspension together with 20 m1 of NE at the chosen final concentrations (10 − 12, 10 − 9, 10 − 7, 10 − 5 and 10 − 3 M) were incubated (37°C and 5% CO2) on culture plates for 30 min, and 20 ml of latex beads (1.09 mm, diluted to 0.5%; SIGMA) were then added. After 30 min incubation, the plates were washed, fixed, and stained, and the number of particles ingested by 100 macrophages was determined (phagocytic index). The results were also expressed as percentage of phagocytosis (percentage of macrophages with phagocytic capacity) and phagocytic efficiency: Phagocytosis index/percentage of phagocytosis =number of latex beads phagocytosed by each active macrophage (Ortega et al., 1993b, 1996). Oxygen-dependent microbicide capacity of macrophages was evaluated by means of superoxide anion production. The assay was performed by the nitroblue tetrazolium (NBT) reduction test. Aliquots of 230 ml of macrophage suspension were preincubated (at 37°C in a bath) with 20 ml of NE at the chosen final concentrations (10 − 12, 10 − 9, 10 − 7, 10 − 5 and 10 − 3 M) for 15 min. Then, 250 ml of preincubated macrophages were mixed with 250 ml of NBT (SIGMA; 1 mg/ml in PBS solution) and 25 ml of latex beads (1%) (stimulated samples) or 25 ml of PBS (nonstimulated samples). After 30 min incubation, the reaction was stopped with HCl (0.5 M) and the samples were centrifuged. The extracellular concentration of superoxide anion (reduced NBT) was then evaluated in the supernatants. In parallel, the intracellular reduced NBT (intracellular concentration of superoxide anion) was extracted with dioxan (SIGMA). The absorbances of the supernatants at 525 nm were determined using HCl or dioxan, respectively, as blanks.
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2.4. Statistical study Data were analysed using the non-parametric ANOVA–Scheffe F-test for comparison of the differences among the results obtained with the different concentrations of norepinephrine. The Student’s t-test was used to evaluate the statistical significance of the variations between the results obtained with each concentration of norepinephrine with respect to the control values. PB 0.05 was taken as the minimum significance level.
3. Results The results regarding the influence of age on the effect of different concentrations of norepinephrine on the phagocytic capacity of macrophages are shown in Fig. 1 and Fig. 2. Incubation of macrophages with 10 − 7 M of NE provoked an increase (PB 0.05) in the phagocytic index in 12- and 48-weeks-old mice, but no change was detected with this concentration in macrophages from 22- and 72-week-old mice. In addition 10 − 3 M of NE increased phagocytosis of macrophages from 22-, 48- and 72-weekold animals, but no significant effect was observed on macrophages from young (12 weeks) mice. Finally, while 10 − 5 M of NE induced a greater phagocytic capacity of macrophages in adult (22 weeks) mice, this concentration decreased it (PB 0.05, using a Student t-test) in 48 weeks-old mice with respect to the control values (Fig. 1). All of these changes were due, in general, to changes in the phagocytic efficiency
Fig. 1. Effect of aging on the modulation induced by norepinephrine on the phagocytic capacity for latex beads of macrophages. Each column is the mean 9 S.D. of 10 experiments performed in duplicate. a, P B0.05 with respect to control values; b, PB 0.05 with respect to 10 − 12 M values; c, P B0.05 with respect to 10 − 9 M values; d, PB 0.05 with respect to 10 − 7 M values; e, PB0.05 with respect to 10 − 5 M values; + , PB 0.05 with respect to control values (Student-t-test).
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Fig. 2. Effect of aging on the modulation induced by norepinephrine on the phagocytic efficiency of macrophages. Each column is the mean 9 S.D. of 10 experiments performed in duplicate. a, P B0.05 with respect to control values; b, PB 0.05 with respect to 10 − 12 M values; c, PB 0.05 with respect to 10 − 9 M values; d, PB 0.05 with respect to 10 − 7 M values; e, PB 0.05 with respect to 10 − 5 M values; +, P B0.05 with respect to control values (Student-t-test).
of macrophages (Fig. 2) and not to changes in the percentage of macrophages; with phagocytic capacity (989 2) (see statistical study in the corresponding figures). Results corresponding to changes with aging in the O− generation by 2 macrophages induced by NE are shown in Table 1 (extracellular levels) and Table 2 (intracellular levels). With respect to extracellular levels of O− 2 in non-stimulated samples (in absence of phagocytosis) NE did not induce any changes in macrophages from 12-week-old mice but we found, in general, an increase induced by NE in the extracellular O− 2 levels of macrophages from 22-, 48- and 72-week-old animals, although the differences (ANOVA) were only significant in adult mice, and after incubation with 10 − 3 M in mature (48 weeks) mice. After phagocytosis of latex beads (stimulated samples), only after incubation with 10 − 7 M of NE the macrophages of young mice showed increased levels of extracellular O− 2 (Table 1). However, in general, no significant changes (ANOVA statistical study) were seen in the effects of NE on the intracellular O− 2 levels in macrophages, both in the absence or in the presence of previous phagocytosis.
4. Discussion In recent years, much interest has centered on the interactions between the nervous system and the immune system. Intact neuroendocrine –immune interactions are essential for the development and functional maintenance of both system (Fabris, 1992). However, it has been indicated that during old age individuals
0.003 90.002 0.009b 90.002 0.007b 90.003 0.009b 90.003 0.008b 90.002 0.009b 90.003
0.010 90.004 0.012 9 0.004 0.011 9 0.04 0.015b 90.005 0.012 90.003 0.011 9 0.004
0.0890.003 0.0089 0.003 0.0089 0.003 0.0079 0.005 0.0089 0.004 0.0099 0.003
NE
E
NE 0.008 9 0.004 0.009 90.003 0.011 90.003 0.012 90.008 0.008 90.004 0.008 90.003
E
0.006 90.004 0.0099 0.003 0.0099 0.004 0.010 90.004 0.009 9 0.003 0.011b 9 0.003
NE
48 weeks
0.01190.003 0.0119 0.004 0.0129 0.005 0.0129 0.004 0.01390.005 0.0139 0.004
E
0.0089 0.003 0.012b 9 0.003 0.009c 9 0.003 0.0119 0.003 0.01190.003 0.0109 0.003
NE
72 weeks
b
a
Each value is the mean 9 S.D. of 10 experiments performed in duplicate N.E., non-stimulated samples; E, stimulated samples. PB0.05 with respect to control values. c PB0.05 with respect to 10−12 M values.
Control 10−12 M 10−9 M 10−7 M 10−5 M 10−3 M
22 weeks
12 weeks
Age of mice (weeks)
0.0109 0.001 0.0109 0.002 0.010 90.002 0.0119 0.003 0.010 90.003 0.0109 0.003
E
Table 1 Effect of aging on the modulation induced by norepinephrine on the extracellular superoxide anion levels (absorbance at 525 nm) in macrophagesa
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0.009 90.003 0.009 90.004 0.010 90.003 0.009 9 0.003 0.008 9 0.003 0.009 9 0.003
0.012 9 0.004 0.012 9 0.004 0.018 90.011 0.012 90.002 0.010 9 0.006 0.012 9 0.007
0.0039 0.001 0.0059 0.001 0.00390.001 0.00590.002 0.0059 0.003 0.00690.003
NE
E
NE 0.037 9 0.025 0.048 9 0.017 0.033 9 0.021 0.055 9 0.034 0.062 9 0.038 0.024 9 0.008
E
0.013 9 0.010 0.015 9 0.005 0.013 90.007 0.013 9 0.005 0.013 90.004 0.013 9 0.004
NE
48 weeks
0.10590.069 0.49090.508 0.479b 9 0.353 0.1979 0.346 0.553b 9 0.303 0.4239 0.339
E
0.021 90.006 0.016 90.002 0.016 90.003 0.016 9 0.002 0.01790.003 0.019 90.005
NE
72 weeks
b
a
Each value is the mean 9 S.D. of 10 experiments performed in duplicate N.E., non-stimulated samples; E, stimulated samples. PB0.05 with respect to control values (Student-t test). c PB0.05 with respect to 10−7 M values.
Control 10−12 M 10−9 M 10−7 M 10−5 M 10−3 M
22 weeks
12 weeks
Age of mice (weeks)
0.290 90.241 0.193 90.157 0.350 9 0.149 0.152 9 0.190 0.486c 9 0.323 0.367 9 0.257
E
Table 2 Effect of aging on the modulation induced by norepinephrine on the intracellular superoxide anion levels (absorbance at 525 nm) in macrophagesa
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present an alteration of the regulatory systems, including a negative effect on neuroimmuneendocrine integration. Moreover, the performance of the regulatory systems is also relevant in stress situations, in which these systems communicate and respond. In fact, stress and aging represent two important models to point out the relevance of interactions between the regulatory systems (Dellwo and Beauchene, 1990; Savastano et al., 1994). Phagocytic cells constitute the first line of defence of the organism against external agression when pathogenic agents have overcome its ‘natural barriers’, such as the skin and mucous membranes. The macrophages are the main phagocytic cells in the tissues, and carry out their non-specific defence function through phagocytosis. Immune function decline is a frequent accompaniment of aging (Makinodan and Kay, 1980) and of stress (Dohms and Metz, 1991), but in contrast to lymphocyte function, it has been reported that both aging (Heidrick, 1972; Ortega et al., 1993a,b) and stress (Dantzer and Kelley, 1989; Ortega et al., 1993b; Forner et al., 1994; Zwilling, 1994) do not cause modifications or enhance the phagocytic activity of macrophages. As found in previous investigations, we have observed that phagocytosis of macrophages decline from young to adult mice, and reaches the highest values in the old mice. This is in agreement with the idea that while the more highly differentiated functions of macrophages, requiring cell-to cell signaling, would be adversely affected by age, the less highly differentiated macrophage functions, such as phagocytosis, are more active in the aged (Kay, 1996). The key question is whether the phagocytic response of macrophages to NE depends on the age of the individuals. NE is quickly released in stress situations, and influences the functional capacity of immune cells, since these cells, including macrophages, have receptors for catecholamines (Landmann et al., 1984; Madden et al., 1995). As indicated for stress in general, most studies have reported that catecholamines decrease the functional capacity of lymphocytes, although their effects could be different depending on the concentration of these simpatetic neurotransmitters (Madden and Livnat, 1991). However, little is known about the non-specific response of macrophages. The results reported in the present work show that the phagocytosis of macrophages may also be modulated by different concentrations of catecholamines. Although we have found a slight decrease induced by some concentrations of NE (10 − 12 M in the adult animals; 10 − 9 and 10 − 5 M in the mature) the main effect of this neurotransmitter on phagocytosis of macrophages is stimulatory. This agrees with the results obtained by other researchers, who found that NE increases E. coli and sheep red blood cells phagocytosis in chicken macrophages (Ali et al., 1994). As mentioned above, other physiological states in which lymphocytes and phagocytic cells respond in different fashion are the stress situations mediated by catecholamines, in which the stimulated phagocytosis of macrophages could counteract the decreased lymphoid activity. Further, it has been reported that other stress hormones, such as corticosterone and prolactin, can also stimulate the phagocytic function of peritoneal macrophages from mice (Ortega et al., 1997). In our opinion the most physiologically relevant results presented here are that the effects induced by NE on the phagocytosis of macrophages differ depending on
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the age of animals. In general, the behaviour of macrophages in response to NE was similar in young (12-week-old) and mature (48-week-old) animals; with an increase in the phagocytic index after incubation with 10 − 7 M of NE, although in mature mice the decrease after incubation with 10 − 5 M and the increase after incubation with 10 − 3 M in the phagocytic index was more marked, with statistically significant differences. The effect found in 22-week-old mice was different, with an increased phagocytic response after incubation with the highest concentrations of NE (10 − 5 and 10 − 3 M). Finally, the modulating effect of NE on macrophages is lower in old animals, in which an increased function was only found after incubation of the macrophages with a concentration of 10 − 3 M. This could mean that, with aging, macrophages lose their response capacity to low concentrations of catecholamines, which agrees with the idea that a dysregulation of immune homeostasis leads to increased vulnerability during stress (Solomon and Benton, 1994). However, the above could be linked to the higher phagocytic capacity of the macrophages from old mice with respect to those from the young, since both the control values and the values obtained after incubation with all NE concentrations evaluated were higher in the macrophages from old mice than those from young, adult or mature animals. The problem is that it may be more important the ‘flexibility’ in the response to changes in simpatetic neurotransmitter than a high response. Changes in phagocytosis can be due to changes in the number of cells with phagocytic capacity, to an altered efficiency of phagocytosis in these cells or to both. In relation to this we found that the changes with aging in the modulation of phagocytosis of macrophages by NE were due to changes in their intrinsic efficiency for phagocytosis. Once the pathogen has been ingested, the next step is its destruction. This process is mediated by a range of destructive granule enzymes and a series of reactive oxidants that are generated during the respiratory burst via activation of NADPH oxidase complexes (Edwards and Watson, 1995). Activated macrophages kill antigenic targets by oxidative pathways including the syntesis of reactive oxygen species (Alvarez et al., 1995). NBT is reduced by the superoxide anion which is produced in a reaction triggered by a membrane oxidase that uses O2 and NADPH as substrate (Rossi, 1986). The superoxide anion is increased during the phagocytic process and it participates in microbicidal events and therefore stimulated samples (after phagocytosis of latex particles) show higher levels of O− 2 than non-stimulated samples. As observed in previous studies performed in our laboratory we have found an age-related increase in O− 2 levels. In the present study we have found, in addition, that in the absence of phagocytosis (non-stimulated samples) NE only induces an increase in the extracellular O− 2 level in macrophages from adult mice. In the absence of phagocytosis, NBT reduction can be considered as a measure of basal metabolism (De la Fuente, 1985). It seems that NE induces an increase in the basal metabolism of macrophages only in adult animals but not at other ages. After phagocytosis, an increased NBT reduction indicates a greater oxygen-dependent microbicidal processes. In this investigation we found that 10 − 7 M of NE induced a significant increase in the extracellular O− 2 level after phagocytosis, and therefore an increased
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microbicidal activity of macrophages from young mice. This agrees with the observed increase in the phagocytosis of latex beads after incubation of macrophages from young mice with 10 − 7 M of NE. However, no modifications were found at this age in the intracellular O− 2 levels. At other ages (22, 48 and 72 weeks) no statistically significant differences induced by NE were obtained, but it seems clear that after incubation with this neurotransmitter macrophages present higher intracellular O− 2 levels after phagocytosis (particularly in 48-week-old animals) although with great differences among animals. It is possible that NE increases the microbicidal activity of macrophages at these ages, which agrees with results obtained by others researchers who have indicated that epinephrine and norepinephrine stimulate the antimycobacterial activity of murine peritoneal macrophages (Miles et al., 1996). In summary, NE can stimulates the phagocytic function of macrophages, above all at high concentrations. This results agrees with those obtained by other authors who found that an increase in the simpatetic activity and in the concentrations of NE increase the phagocytosis and citotoxicity of these cells (Hartmann et al., 1987). It seems that the effects of catecholamines on the immune system are very complex and some times contradictory, depending on the concentration present in the immune cells. Thus, some researchers have postulated that catecholamines acts as negative modulators of the immune activity of macrophages, and others have shown that macrophages increase various aspects of their function because of increasing concentrations of NE. In this work it is also shown that the modulation of the non specific function of macrophages by NE is also related to the age of animals. Apparently, during old age higher concentrations of NE are needed for stimulation of the phagocytic function of macrophages.
Acknowledgements This work was supported by grants from the ‘Fondo de Investigacio´n Sanitaria’ (F.I.S. 96/1059 and 99/0815) and ‘Junta de Extremadura-Fondo Social Europeo’.
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Changes with aging in the modulation of macrophages by norepinephrine E. Ortega a,*, J.J. Garcı´a a, M.C. Sa´ez a, M. De la Fuente b a
Department of Physiology, Faculty of Science, Uni6ersity of Extremadura, A6enida de El6a s/n, 06071 Badajoz, Spain b Department of Animal Biology (Animal Physiology), Facutly of Biological Sciences, Complutense Uni6ersity, 28040 Madrid, Spain Received 8 March 2000; received in revised form 28 May 2000; accepted 29 June 2000
Abstract The effect of aging on the norepinephrine (NE)-induced modulation of phagocytic and oxygen-dependent microbicidal processes of mouse peritoneal macrophages was studied. Phagocytosis of latex beads on culture plates and superoxide anion production was evaluated in young (12 weeks), adult (22 weeks), mature (48 weeks) and old (72 weeks) BALB/c mice after in vitro incubation with 10 − 12, 10 − 9, 10 − 7, 10 − 5 or 10 − 3 M concentrations of NE. The results indicate that the phagocytic response to NE is quite similar in young and mature mice, with increased phagocytosis after incubation with 10 − 7 and 10 − 3 M, and decreased phagocytosis with 10 − 5 M. Macrophages from adult mice increased their phagocytic capacity after incubation with the highest concentrations of NE (10 − 5 and 10 − 3 M) and macrophages from old animals only were stimulated with 10 − 3 M. In addition, it was found that usually NE increased the extracellular superoxide anion production in the absence of phagocytosis in adult mice. No statistically significant changes were found in intracellular superoxide anion levels, but an increase was seen after phagocytosis in macrophages from adult, mature and old animals, especially after incubation with 10 − 5 M. In conclusion, the results obtained indicate that the modulation of macrophages by NE does not only depend on the concentration of this neurotransmitter, but also on age. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; Macrophages; Norepinephrine; Mice
* Corresponding author. Fax: +34-24-271304. E-mail address: [email protected] (E. Ortega). 0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 6 0 - 3
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1. Introduction It is well-known that the activity of the immune system declines with age. Clinical evidence for immunosenescence is the high incidence of tumours in elderly people. Also significant is the greater susceptibility of the aged to infections from pathogens to which they had been relatively resistant early in life (Weigle, 1989). In fact, elderly subjects who keep their immune functions at an excepcionally high level, live longer and may even become centenarians (Hirokawa, 1998). In some cases, the impairment of the immune response with age is a result of defective T-cell and/or B-cell functions. The T-cell functions especially show a marked decline with age, which is related to the involution of the thymus (Hirokawa, 1998; Solana et al., 1991; Utsuyama et al., 1991; Kay, 1996). Moreover, macrophages must be also taken into account as part of the cellular collaboration established in the antigenic response. A dysfunction of these cells could also contribute to declining lymphocyte function in old age (Callard, 1978; Makinodan and Kay, 1980; Solana et al., 1991). Thus, macrophages from old mice secrete inadequate amounts of IL-1, leading to decreased stimulation of T-helper cells and thereby contribute to diminished IL-2 production (Solomon and Benton, 1994). However, some studies suggest that the macrophage function, unlike the lymphocyte function, does not decline with age (Makinodan and Kay, 1980; Sondell et al., 1990), and there has even been observed a stimulation of the capacity of the monocyte–macrophage system in old individuals (Heidrick, 1972; Finger et al., 1982; Kuroiwa et al., 1989; Ortega et al., 1993a), especially with respect to phagocytosis. A similar situation is also found in stress, which also stimulates the phagocytosis of macrophages; and, on the contrary, causes a decline in lymphocyte function. Indeed, phagocytes can be stimulated by stress hormones (Cupps and Fauci, 1982; Dantzer and Kelley, 1989; Dohms and Metz, 1991; Forner et al., 1994; Ortega et al., 1997). In addition, during old age individuals present an alteration of the regulatory systems, including a negative effect on neuroimmune –endocrine integration (Fabris, 1992). It has been reported that this dysregulation of immune homeostasis, leading to vulnerability and failure under stress, may be linked to changes in the cellular milieu in which neurons and immune and endocrine cells function (Solomon and Benton, 1994), and can contribute to immunosenescence. Macrophages show receptors for catecholamines, and it has been reported that macrophage function is modified by catecholamines and adrenergic agonists (Madden and Livnat, 1991). However while an immunossupresor effect of catecholamines on lymphocytes has been established (Madden and Livnat, 1991), an immmunomodulation of these sympatetic mediators for non-specific immune responses of macrophages is uncertain, and probably it would be related to the age of individuals. In view of the above, the purpose of this investigation was to study whether the response of macrophages to norepinephrine is modified by aging.
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2. Materials and methods
2.1. Animals We have used in our study young (12 weeks), adult (22 weeks), mature (48 weeks) and old (72 weeks) BALB/c mice (Mus musculus), maintained at a constant temperature (22°9 2°C) on a 12-h light/dark cycle and standard laboratory diet with free access to food and water. The mice were examined, and only those without signs of malignancy or other pathological manifestations were used in the experiments.
2.2. Collection of cells The abdomen was cleansed with 70% ethanol, the abdominal skin carefully dissected without opening the peritoneum, and 3 ml of Hank’s solution adjusted to pH 7.4 injected i.p. The abdomen was massaged and the peritoneal exudate cells were removed, with recovery of 90–95% of the injected volume of fluid. The cells (macrophages and lymphocytes) were counted and adjusted to a final concentration of 5× 105 macrophages per ml in Hank’s solution (Ortega et al., 1993b).
2.3. Effect of norepinephrine (NE) on phagocytic function of macrophages In order to study the effect of NE on the ingestion (phagocytosis) capacity for latex beads of peritoneal macrophages, 200 ml of macrophage suspension together with 20 m1 of NE at the chosen final concentrations (10 − 12, 10 − 9, 10 − 7, 10 − 5 and 10 − 3 M) were incubated (37°C and 5% CO2) on culture plates for 30 min, and 20 ml of latex beads (1.09 mm, diluted to 0.5%; SIGMA) were then added. After 30 min incubation, the plates were washed, fixed, and stained, and the number of particles ingested by 100 macrophages was determined (phagocytic index). The results were also expressed as percentage of phagocytosis (percentage of macrophages with phagocytic capacity) and phagocytic efficiency: Phagocytosis index/percentage of phagocytosis =number of latex beads phagocytosed by each active macrophage (Ortega et al., 1993b, 1996). Oxygen-dependent microbicide capacity of macrophages was evaluated by means of superoxide anion production. The assay was performed by the nitroblue tetrazolium (NBT) reduction test. Aliquots of 230 ml of macrophage suspension were preincubated (at 37°C in a bath) with 20 ml of NE at the chosen final concentrations (10 − 12, 10 − 9, 10 − 7, 10 − 5 and 10 − 3 M) for 15 min. Then, 250 ml of preincubated macrophages were mixed with 250 ml of NBT (SIGMA; 1 mg/ml in PBS solution) and 25 ml of latex beads (1%) (stimulated samples) or 25 ml of PBS (nonstimulated samples). After 30 min incubation, the reaction was stopped with HCl (0.5 M) and the samples were centrifuged. The extracellular concentration of superoxide anion (reduced NBT) was then evaluated in the supernatants. In parallel, the intracellular reduced NBT (intracellular concentration of superoxide anion) was extracted with dioxan (SIGMA). The absorbances of the supernatants at 525 nm were determined using HCl or dioxan, respectively, as blanks.
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2.4. Statistical study Data were analysed using the non-parametric ANOVA–Scheffe F-test for comparison of the differences among the results obtained with the different concentrations of norepinephrine. The Student’s t-test was used to evaluate the statistical significance of the variations between the results obtained with each concentration of norepinephrine with respect to the control values. PB 0.05 was taken as the minimum significance level.
3. Results The results regarding the influence of age on the effect of different concentrations of norepinephrine on the phagocytic capacity of macrophages are shown in Fig. 1 and Fig. 2. Incubation of macrophages with 10 − 7 M of NE provoked an increase (PB 0.05) in the phagocytic index in 12- and 48-weeks-old mice, but no change was detected with this concentration in macrophages from 22- and 72-week-old mice. In addition 10 − 3 M of NE increased phagocytosis of macrophages from 22-, 48- and 72-weekold animals, but no significant effect was observed on macrophages from young (12 weeks) mice. Finally, while 10 − 5 M of NE induced a greater phagocytic capacity of macrophages in adult (22 weeks) mice, this concentration decreased it (PB 0.05, using a Student t-test) in 48 weeks-old mice with respect to the control values (Fig. 1). All of these changes were due, in general, to changes in the phagocytic efficiency
Fig. 1. Effect of aging on the modulation induced by norepinephrine on the phagocytic capacity for latex beads of macrophages. Each column is the mean 9 S.D. of 10 experiments performed in duplicate. a, P B0.05 with respect to control values; b, PB 0.05 with respect to 10 − 12 M values; c, P B0.05 with respect to 10 − 9 M values; d, PB 0.05 with respect to 10 − 7 M values; e, PB0.05 with respect to 10 − 5 M values; + , PB 0.05 with respect to control values (Student-t-test).
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Fig. 2. Effect of aging on the modulation induced by norepinephrine on the phagocytic efficiency of macrophages. Each column is the mean 9 S.D. of 10 experiments performed in duplicate. a, P B0.05 with respect to control values; b, PB 0.05 with respect to 10 − 12 M values; c, PB 0.05 with respect to 10 − 9 M values; d, PB 0.05 with respect to 10 − 7 M values; e, PB 0.05 with respect to 10 − 5 M values; +, P B0.05 with respect to control values (Student-t-test).
of macrophages (Fig. 2) and not to changes in the percentage of macrophages; with phagocytic capacity (989 2) (see statistical study in the corresponding figures). Results corresponding to changes with aging in the O− generation by 2 macrophages induced by NE are shown in Table 1 (extracellular levels) and Table 2 (intracellular levels). With respect to extracellular levels of O− 2 in non-stimulated samples (in absence of phagocytosis) NE did not induce any changes in macrophages from 12-week-old mice but we found, in general, an increase induced by NE in the extracellular O− 2 levels of macrophages from 22-, 48- and 72-week-old animals, although the differences (ANOVA) were only significant in adult mice, and after incubation with 10 − 3 M in mature (48 weeks) mice. After phagocytosis of latex beads (stimulated samples), only after incubation with 10 − 7 M of NE the macrophages of young mice showed increased levels of extracellular O− 2 (Table 1). However, in general, no significant changes (ANOVA statistical study) were seen in the effects of NE on the intracellular O− 2 levels in macrophages, both in the absence or in the presence of previous phagocytosis.
4. Discussion In recent years, much interest has centered on the interactions between the nervous system and the immune system. Intact neuroendocrine –immune interactions are essential for the development and functional maintenance of both system (Fabris, 1992). However, it has been indicated that during old age individuals
0.003 90.002 0.009b 90.002 0.007b 90.003 0.009b 90.003 0.008b 90.002 0.009b 90.003
0.010 90.004 0.012 9 0.004 0.011 9 0.04 0.015b 90.005 0.012 90.003 0.011 9 0.004
0.0890.003 0.0089 0.003 0.0089 0.003 0.0079 0.005 0.0089 0.004 0.0099 0.003
NE
E
NE 0.008 9 0.004 0.009 90.003 0.011 90.003 0.012 90.008 0.008 90.004 0.008 90.003
E
0.006 90.004 0.0099 0.003 0.0099 0.004 0.010 90.004 0.009 9 0.003 0.011b 9 0.003
NE
48 weeks
0.01190.003 0.0119 0.004 0.0129 0.005 0.0129 0.004 0.01390.005 0.0139 0.004
E
0.0089 0.003 0.012b 9 0.003 0.009c 9 0.003 0.0119 0.003 0.01190.003 0.0109 0.003
NE
72 weeks
b
a
Each value is the mean 9 S.D. of 10 experiments performed in duplicate N.E., non-stimulated samples; E, stimulated samples. PB0.05 with respect to control values. c PB0.05 with respect to 10−12 M values.
Control 10−12 M 10−9 M 10−7 M 10−5 M 10−3 M
22 weeks
12 weeks
Age of mice (weeks)
0.0109 0.001 0.0109 0.002 0.010 90.002 0.0119 0.003 0.010 90.003 0.0109 0.003
E
Table 1 Effect of aging on the modulation induced by norepinephrine on the extracellular superoxide anion levels (absorbance at 525 nm) in macrophagesa
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0.009 90.003 0.009 90.004 0.010 90.003 0.009 9 0.003 0.008 9 0.003 0.009 9 0.003
0.012 9 0.004 0.012 9 0.004 0.018 90.011 0.012 90.002 0.010 9 0.006 0.012 9 0.007
0.0039 0.001 0.0059 0.001 0.00390.001 0.00590.002 0.0059 0.003 0.00690.003
NE
E
NE 0.037 9 0.025 0.048 9 0.017 0.033 9 0.021 0.055 9 0.034 0.062 9 0.038 0.024 9 0.008
E
0.013 9 0.010 0.015 9 0.005 0.013 90.007 0.013 9 0.005 0.013 90.004 0.013 9 0.004
NE
48 weeks
0.10590.069 0.49090.508 0.479b 9 0.353 0.1979 0.346 0.553b 9 0.303 0.4239 0.339
E
0.021 90.006 0.016 90.002 0.016 90.003 0.016 9 0.002 0.01790.003 0.019 90.005
NE
72 weeks
b
a
Each value is the mean 9 S.D. of 10 experiments performed in duplicate N.E., non-stimulated samples; E, stimulated samples. PB0.05 with respect to control values (Student-t test). c PB0.05 with respect to 10−7 M values.
Control 10−12 M 10−9 M 10−7 M 10−5 M 10−3 M
22 weeks
12 weeks
Age of mice (weeks)
0.290 90.241 0.193 90.157 0.350 9 0.149 0.152 9 0.190 0.486c 9 0.323 0.367 9 0.257
E
Table 2 Effect of aging on the modulation induced by norepinephrine on the intracellular superoxide anion levels (absorbance at 525 nm) in macrophagesa
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present an alteration of the regulatory systems, including a negative effect on neuroimmuneendocrine integration. Moreover, the performance of the regulatory systems is also relevant in stress situations, in which these systems communicate and respond. In fact, stress and aging represent two important models to point out the relevance of interactions between the regulatory systems (Dellwo and Beauchene, 1990; Savastano et al., 1994). Phagocytic cells constitute the first line of defence of the organism against external agression when pathogenic agents have overcome its ‘natural barriers’, such as the skin and mucous membranes. The macrophages are the main phagocytic cells in the tissues, and carry out their non-specific defence function through phagocytosis. Immune function decline is a frequent accompaniment of aging (Makinodan and Kay, 1980) and of stress (Dohms and Metz, 1991), but in contrast to lymphocyte function, it has been reported that both aging (Heidrick, 1972; Ortega et al., 1993a,b) and stress (Dantzer and Kelley, 1989; Ortega et al., 1993b; Forner et al., 1994; Zwilling, 1994) do not cause modifications or enhance the phagocytic activity of macrophages. As found in previous investigations, we have observed that phagocytosis of macrophages decline from young to adult mice, and reaches the highest values in the old mice. This is in agreement with the idea that while the more highly differentiated functions of macrophages, requiring cell-to cell signaling, would be adversely affected by age, the less highly differentiated macrophage functions, such as phagocytosis, are more active in the aged (Kay, 1996). The key question is whether the phagocytic response of macrophages to NE depends on the age of the individuals. NE is quickly released in stress situations, and influences the functional capacity of immune cells, since these cells, including macrophages, have receptors for catecholamines (Landmann et al., 1984; Madden et al., 1995). As indicated for stress in general, most studies have reported that catecholamines decrease the functional capacity of lymphocytes, although their effects could be different depending on the concentration of these simpatetic neurotransmitters (Madden and Livnat, 1991). However, little is known about the non-specific response of macrophages. The results reported in the present work show that the phagocytosis of macrophages may also be modulated by different concentrations of catecholamines. Although we have found a slight decrease induced by some concentrations of NE (10 − 12 M in the adult animals; 10 − 9 and 10 − 5 M in the mature) the main effect of this neurotransmitter on phagocytosis of macrophages is stimulatory. This agrees with the results obtained by other researchers, who found that NE increases E. coli and sheep red blood cells phagocytosis in chicken macrophages (Ali et al., 1994). As mentioned above, other physiological states in which lymphocytes and phagocytic cells respond in different fashion are the stress situations mediated by catecholamines, in which the stimulated phagocytosis of macrophages could counteract the decreased lymphoid activity. Further, it has been reported that other stress hormones, such as corticosterone and prolactin, can also stimulate the phagocytic function of peritoneal macrophages from mice (Ortega et al., 1997). In our opinion the most physiologically relevant results presented here are that the effects induced by NE on the phagocytosis of macrophages differ depending on
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the age of animals. In general, the behaviour of macrophages in response to NE was similar in young (12-week-old) and mature (48-week-old) animals; with an increase in the phagocytic index after incubation with 10 − 7 M of NE, although in mature mice the decrease after incubation with 10 − 5 M and the increase after incubation with 10 − 3 M in the phagocytic index was more marked, with statistically significant differences. The effect found in 22-week-old mice was different, with an increased phagocytic response after incubation with the highest concentrations of NE (10 − 5 and 10 − 3 M). Finally, the modulating effect of NE on macrophages is lower in old animals, in which an increased function was only found after incubation of the macrophages with a concentration of 10 − 3 M. This could mean that, with aging, macrophages lose their response capacity to low concentrations of catecholamines, which agrees with the idea that a dysregulation of immune homeostasis leads to increased vulnerability during stress (Solomon and Benton, 1994). However, the above could be linked to the higher phagocytic capacity of the macrophages from old mice with respect to those from the young, since both the control values and the values obtained after incubation with all NE concentrations evaluated were higher in the macrophages from old mice than those from young, adult or mature animals. The problem is that it may be more important the ‘flexibility’ in the response to changes in simpatetic neurotransmitter than a high response. Changes in phagocytosis can be due to changes in the number of cells with phagocytic capacity, to an altered efficiency of phagocytosis in these cells or to both. In relation to this we found that the changes with aging in the modulation of phagocytosis of macrophages by NE were due to changes in their intrinsic efficiency for phagocytosis. Once the pathogen has been ingested, the next step is its destruction. This process is mediated by a range of destructive granule enzymes and a series of reactive oxidants that are generated during the respiratory burst via activation of NADPH oxidase complexes (Edwards and Watson, 1995). Activated macrophages kill antigenic targets by oxidative pathways including the syntesis of reactive oxygen species (Alvarez et al., 1995). NBT is reduced by the superoxide anion which is produced in a reaction triggered by a membrane oxidase that uses O2 and NADPH as substrate (Rossi, 1986). The superoxide anion is increased during the phagocytic process and it participates in microbicidal events and therefore stimulated samples (after phagocytosis of latex particles) show higher levels of O− 2 than non-stimulated samples. As observed in previous studies performed in our laboratory we have found an age-related increase in O− 2 levels. In the present study we have found, in addition, that in the absence of phagocytosis (non-stimulated samples) NE only induces an increase in the extracellular O− 2 level in macrophages from adult mice. In the absence of phagocytosis, NBT reduction can be considered as a measure of basal metabolism (De la Fuente, 1985). It seems that NE induces an increase in the basal metabolism of macrophages only in adult animals but not at other ages. After phagocytosis, an increased NBT reduction indicates a greater oxygen-dependent microbicidal processes. In this investigation we found that 10 − 7 M of NE induced a significant increase in the extracellular O− 2 level after phagocytosis, and therefore an increased
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microbicidal activity of macrophages from young mice. This agrees with the observed increase in the phagocytosis of latex beads after incubation of macrophages from young mice with 10 − 7 M of NE. However, no modifications were found at this age in the intracellular O− 2 levels. At other ages (22, 48 and 72 weeks) no statistically significant differences induced by NE were obtained, but it seems clear that after incubation with this neurotransmitter macrophages present higher intracellular O− 2 levels after phagocytosis (particularly in 48-week-old animals) although with great differences among animals. It is possible that NE increases the microbicidal activity of macrophages at these ages, which agrees with results obtained by others researchers who have indicated that epinephrine and norepinephrine stimulate the antimycobacterial activity of murine peritoneal macrophages (Miles et al., 1996). In summary, NE can stimulates the phagocytic function of macrophages, above all at high concentrations. This results agrees with those obtained by other authors who found that an increase in the simpatetic activity and in the concentrations of NE increase the phagocytosis and citotoxicity of these cells (Hartmann et al., 1987). It seems that the effects of catecholamines on the immune system are very complex and some times contradictory, depending on the concentration present in the immune cells. Thus, some researchers have postulated that catecholamines acts as negative modulators of the immune activity of macrophages, and others have shown that macrophages increase various aspects of their function because of increasing concentrations of NE. In this work it is also shown that the modulation of the non specific function of macrophages by NE is also related to the age of animals. Apparently, during old age higher concentrations of NE are needed for stimulation of the phagocytic function of macrophages.
Acknowledgements This work was supported by grants from the ‘Fondo de Investigacio´n Sanitaria’ (F.I.S. 96/1059 and 99/0815) and ‘Junta de Extremadura-Fondo Social Europeo’.
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