Changes with aging in the modulation by neuropeptide Y of murine peritoneal macrophage functions

Journal of Neuroimmunology 116 Ž2001. 156–167 www.elsevier.comrlocaterjneuroin

Changes with aging in the modulation by neuropeptide Y of murine peritoneal macrophage functions M. De la Fuente ) , M. Del Rıo, ´ S. Medina Department of Animal Physiology, Biology Faculty, Complutense UniÕersity of Madrid, Madrid, Spain Received 27 November 2000; received in revised form 7 March 2001; accepted 7 March 2001

Abstract Some age-related changes in immune function may be due, at least in part, to a disturbance in the communication between the nervous and immune systems. In the present work, the effects in vitro of neuropeptide Y ŽNPY. Ž10y13 to 10y7 M. on different peritoneal macrophage functions Žadherence to substrate, chemotaxis, phagocytosis, superoxide anion production, and the release of TNFa and IL-1b . have been studied on cells from young Ž12 " 2 weeks., adult Ž24 " 2 weeks., mature Ž50 " 2 weeks. and old Ž72 " 2 weeks. BALBrc mice. The specificity of these actions was confirmed using two C-terminal fragments of NPY, and the intracellular messengers Žprotein kinase C and cAMP. involved in the action of the neuropeptide were also analyzed. The results show that the functions studied change with aging and that the effects of NPY on each function, which are carried out through specific receptors, as well as on intracellular pathway, differ depending on age, maintaining the immune functions at physiologically adequate levels in old animals. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Aging; Neuropeptide Y; Macrophage functions; Mice

1. Introduction A great deal of evidence regarding a close communication and interaction between the nervous and immune systems supports the existence of a neuroimmune axis ŽBesedovsky and Del Rey, 1996; De la Fuente, 1999.. Aging is associated with a decline of many physiological functions, including those of the nervous and the immune system, as well as the relationship of these two systems, which leads to a loss of homeostasis ŽFabris, 1991; Goya et al., 1999.. Thus, experimental alteration of one of the two systems can modify or even restore the function of the other ŽFabris, 1991.. In the immune system, a functional impairment with aging has been well established for specific immune responses ŽPawelec, 1999; Ginaldi et al., 1999.; however, the results are not so evident as regards the non-specific immune response ŽOrtega et al., 1993; McArthur, 1998.. ) Corresponding author. Departamento Biologıa ´ Animal II ŽFisiologıa ´ Animal., Facultad de Ciencias Biologicas, Universidad Complutense. Av. ´ Complutense srn, E-28040 Madrid, Spain. Tel.: q34-91-394-4986; fax: q34-91-394-4935. E-mail address: [email protected] ŽM. De la Fuente..

Neuropeptide Y ŽNPY., a peptide from the pancreatic polypeptide ŽPP. family, has an important role as neurotransmitter and neuromodulator in the nervous system and it is released by noradrenergic neurons along with norepinephrine ŽFriedman and Irwin, 1997.. Besides its widespread distribution in both central and peripheral neurons, it has been found in the nerve endings of immunocompetent organs ŽRomano et al., 1991.. Splenic cells have been shown to express receptors for NPY ŽGehlert and Gackenheimer, 1996. and several studies, in vivo and in vitro, have revealed functional changes in immune cells by this neuropeptide. Thus, NPY causes a decrease in the proliferative response of lymphocytes to mitogens ŽSoder ¨ and Hellstrom, ¨ 1987; Medina et al., 2000a. while no change occurs in spontaneous lymphoproliferation ŽJohansson and Sandberg, 1989.. In addition, our group observed a stimulation by NPY of murine peritoneal macrophage functions in adult mice ŽDe la Fuente et al., 1993a. as well as an increment in lymphocyte migration ŽMedina et al., 2000b. and NK activity ŽMedina et al., 1998.. On the other hand, the possible changes in NPY immunomodulation with aging have been scarcely studied. In recent work, we have observed changes with age in the modulation of lymphocyte adherence and mobility ŽMedina et al., 2000b.,

0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 0 1 . 0 0 2 9 7 - 1

M. De la Fuente et al.r Journal of Neuroimmunology 116 (2001) 156–167

lymphoproliferative response ŽMedina et al., 2000a. and NK activity ŽMedina et al., 1998.. The aim of the present work was to study the changes with aging in the macrophage response to NPY, the cell that constitutes the first line of immunological defense of the organism against pathogenic agents and the most important phagocytic cell in the tissues. Moreover, peritoneal macrophages, the cells used in the present study, are representative of other populations of these phagocytes ŽUnanue, 1989.. We show here the changes with aging in the effects of a wide range of concentrations of NPY on several macrophage functions such as adherence to substrate, chemotaxis, phagocytosis and production of superoxide anion. In addition to this study of the different stages of the most representative activity of phagocytes i.e., the phagocytic process, the changes in the release by macrophages of cytokines such as TNFa and IL-1b was also analyzed. Since NPY has been shown to inhibit adenylate cyclase activity in the central nervous system ŽWestlind-Danielsson et al., 1987., to increase intracellular calcium and inositol phosphate concentrations in human erytroleukemia cells ŽDaniels et al., 1989., and to activate protein kinase C ŽPKC. in murine peritoneal macrophages ŽDe la Fuente et al., 1993a., possible changes in the intracellular pathway followed by NPY in murine peritoneal macrophages with aging were also investigated.

157

C-terminal fragments of NPY: NPY Ž18–36. and Dtyr 27,36 D-thr 32 –NPYŽ27–36. from Bachem ŽFeinchemikalien, Switzerland.. The neuropeptides were dissolved in phosphate-buffered saline ŽPBS. solution and PMA was dissolved in dimethylsulfoxide. RPMI 1640 medium and fetal calf serum were obtained from Gibco ŽPaisley, Scotland.. MIF Žmigratory inhibitory factor. plates were obtained from Steriling ŽTeddington, UK.. Trypan blue and Giemsa for plate staining were from Merck ŽGermany.. EGTA and calcium ionophore A23187 were from Boehringer Mannheim ŽGermany.. The other chemicals used were from Merck. 2.3. Collection of peritoneal exudate cells (PECs) Mice were sacrificed by cervical dislocation according to the guidelines of the European Community Council Directives 86r6091 EEC. The peritoneal resident cells, containing macrophages and lymphocytes, were obtained as previously described by De la Fuente et al. Ž1993a.. The macrophages, identified by morphology and non-specific esterase staining, were counted in Neubauer chambers and adjusted at 5 = 10 5 cellsrml of Hank’s solution. Cellular viability, routinely measured before and after each experiment by the trypan blue exclusion test, was higher than 95% in all experiments. All incubations were performed at 378C in a humidified atmosphere of 5% CO 2 . NPY was used at concentrations ranging from 10y1 3 to 10y7 M.

2. Materials and methods 2.4. Assay of adherence capacity 2.1. Animals Female BALBrc mice were purchased from IFFA CREDO and were bred until the ages of 12 " 2 weeks Žyoung., 24 " 2 weeks Žadult., 50 " 2 weeks Žmature. and 72 " 2 weeks Žold.. All animals were maintained at a constant temperature of 22 " 28C in sterile conditions inside an aseptic air negative-pressure environmental cabinet ŽFlufrance, Cachan, France. on a 12-h lightrdark cycle and fed Sander Mus ŽPANLAB. and water ad libitum. Although in previous studies we observed that the oestrous 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. The animals used did not show any sign of malignancy or other pathological processes.

For quantification of substrate adherence capacity, the adherence of macrophages to a smooth plastic surface was determined because it resembles adherence to animal tissues ŽNoga et al., 1984.. Following the method described by De la Fuente et al. Ž1991., aliquots of 200 ml of peritoneal suspension were placed in eppendorf tubes and the NPY, at concentrations ranging from 10y1 3 to 10y7 M, or Hank’s solution Žcontrols. were added. At 10 min of incubation, aliquots of 10 ml from each sample were removed after gently shaking to resuspend the sedimented cells, and the number of non-adhered macrophages was determined using Neubauer chambers and optical microscopy. The changes with aging in the effects of 10y1 1 M NPY have been also studied at 20 and 30 min of incubation. The adherence index was calculated according to the following equation:

2.2. Reagents

AI s Ž 1 y macrophagesrml supernatantsrmacrophages

Neuropeptide Y ŽNPY., gastrin-releasing peptide ŽGRP., eight-sulfated cholecystokinin ŽCCK-8s., phorbol myristate acetate ŽPMA., retinal, adenosine, forskoline, nitroblue tetrazolium ŽNBT., f-Met–Leu–Phe ŽFMLP., trypsin, Concanavalin A, lipopolysaccharide from Escherichia coli ŽLPS., 1,4-dithioerythritol and latex beads Ž1.09 mm diameter. were purchased from Sigma ŽSt. Louis, MA. and

rml original sample. = 100. Calcium ionophore A23187 and FMLP were used as positive and negative controls, respectively ŽDe la Fuente et al., 1993a; Garrido et al., 1996., as well as the neuropeptide GRP Ž10y1 0 M. which has been previously shown to stimulate the adherence of macrophages ŽDe la Fuente et al., 1991..

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2.5. Assay of chemotaxis Chemotaxis was evaluated according to a modification ŽDe la Fuente et al., 1991. of Boyden’s technique ŽBoyden, 1962., which basically consists in the use of chambers with two compartments separated by a filter of 3 mm of pore diameter. Aliquots of 300 ml of the peritoneal suspension were deposited in the upper compartment with 30 ml of different NPY concentrations Ž10y1 3 to 10y7 M. or Hank’s solution in the controls. In the lower compartment, aliquots of 400 ml of the chemoattractant FMLP at the concentration of 10y8 M, were deposited. The chambers were incubated for 3 h, the filters fixed and stained and the number of macrophages in the lower face of the filter were counted using optical microscopy Žimmersion objective.. Calcium ionophore and GRP ŽDe la Fuente et al., 1991, 1993a., as well as retinal and CCK-8s ŽDe la Fuente et al., 1995a; Garrido et al., 1996. were assayed as positive and negative controls, respectively.

tained were expressed as nmol of NBT reduced per 10 6 macrophages by extrapolating from a standard curve of NBT reduced with 1,4-dithioerythritol. PMA and GRP as well as retinal and CCK-8s ŽDe la Fuente et al., 1991, 1995a; Delgado et al., 1996. were also assayed as positive and negative controls, respectively. 2.8. Specificity of the NPY effects The specificity of the effects observed was evaluated adding two C-terminal fragments of NPY, i.e: NPY Ž18– 36. and D-tyr 27,36 D thr 32 –NPYŽ27–36. which act as agonist andror antagonist for Y1 , Y2 , and Y3 receptors ŽBalasubramaniam and Sheriff, 1990; Levite et al., 1998. and as antagonist of Y1 and Y2 receptors ŽMyers et al., 1995., respectively. In the phagocytosis assay both C-terminal fragments of NPY were assayed, whereas in the other phagocytic stages only NPY 18–36 were analyzed since 27,36 32 D-tyr D thr –NPYŽ27–36. showed itself agonist properties in phagocytosis assay.

2.6. Phagocytosis assay Phagocytosis of inert particles Žlatex beads 1.09 mm diluted to 1% in PBS. was carried out following the method previously described ŽDe la Fuente et al., 1991., incubating aliquots of 200 ml of the peritoneal suspension in MIF Žmigratory inhibitory factor. plates for 30 min. The adhered monolayer was washed with PBS at 378C, resuspended in 200 ml of Hank’s solution and incubated with 20 ml of latex Ž1% in PBS., as well as with 20 ml of NPY concentrations Ž10y1 3 to 10y7 M. or Hank’s solution Žcontrols.. After 30 min of incubation, the plates were washed with PBS, fixed and stained, and the number of latex beads ingested per 100 macrophages was counted by optical microscopy. Calcium ionophore and GRP ŽDe la Fuente et al., 1991, 1993a. were used as positive controls whereas EGTA and CCK-8s ŽDe la Fuente et al., 1995a. were assayed as negative ones. 2.7. Superoxide anion production The nitroblue tetrazolium ŽNBT. reduction test, based on an equimolecular reaction between NBT and superoxide anion ŽBagasra et al., 1998., was carried out for determination of superoxide production according to a method previously described ŽDe la Fuente et al., 1991.. Briefly, aliquots of 250 ml of peritoneal suspension were mixed with 250 ml of NBT solution Ž1 mgrml Hank’s solution. and the different concentrations of NPY Ž10y1 3 to 10y7 M. or Hank’s solution Žcontrols.. Aliquots of 50 ml of latex beads were added to one sample set Žstimulated samples. and 50 ml of Hank’s solution to the other set Žnon-stimulated samples.. After 60 min of incubation in a bath at 378C, the reaction was stopped and, following centrifugation, the supernatants were discarded and the reduced NBT was extracted with dioxan. The data ob-

2.9. Implication of cAMP in the effect of NPY on phagocytosis In leukocyte phagocytosis assays, the implication of cAMP in the action of NPY was determined using an adenylate cyclase inhibitor, adenosine, and an activator, forskoline, at 10y7 and 10y5 M concentrations, respectively ŽDelgado et al., 1996.. The levels of intracellular cAMP in isolated peritoneal macrophages from adult and old mice were also measured. Briefly, peritoneal suspensions were dispensed into eppendorf tubes and incubated for 45 min with the addition of trypsin Ž0.5%. in order to remove non-macrophage cells and debris. Then, adhered macrophages were gently washed and incubated with RPMI 1640 medium for up to 24 h. Macrophages obtained as indicated, were incubated with NPY Ž10y1 2 to 10y8 M., forskoline Ž10y5 M., adenosine Ž10y7 M., GRP Ž10y1 0 M. and CCK-8s Ž10y1 0 M. for 30 and 60 s. After incubation, 500 ml of 10% cold trifluoroacetic acid were added to each tube, and after vigorous shaking for 2 min, the samples were centrifuged for 10 min at 300 g. Then, supernatants were collected and lyophilized. Finally, the samples reconstituted with the corresponding assay buffer were subjected to an ELISA from Cayman Chemical, the minimum detectable dose of mouse cAMP being 0.04 pmolrml and a limit of the procedure of up to 3 pmolrml. 2.10. Implication of PKC in the effect of NPY on phagocytosis The implication of protein kinase C ŽPKC. activation in the NPY actions was ascertained adding the activator of this enzyme, PMA Ž50 ngrml., or an inhibitor of PKC, retinal Ž20 mM. ŽDe la Fuente et al., 1993b., to the leukocyte phagocytosis assay.

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2.11. Tumor necrosis factor alpha (TNFa ) release in response to LPS and NPY

mouse IL-1b of 3.0 pgrml and a limit of the procedure of up to 500 pgrml.

TNFa was measured in the supernatants of cultures of adherent cells from the peritoneal suspension from adult and old mice. Cells were incubated following the method previously described ŽVıctor et al., 2000. with Hank’s ´ solution at a final concentration of 2 = 10 5 cellsr200 mlrwell in 96 well plates for 45 min to allow macrophages to form a monolayer. Then, after two washes, RPMI-1640 medium without phenol red and with L-glutamine and 10% calf serum was added together with LPS Ž10 mgrml. alone or with NPY Žfrom 10y1 2 to 10y8 M.. After a 24 h incubation, plates were centrifuged and the TNFa production was quantified in the supernatants using an R & D System ŽMinneapolis, USA. immunoassay with recombinant mouse TNFa , a minimum detectable dose of mouse TNFa of 5.1 pgrml and a limit of the procedure of up to 1500 pgrml.

2.13. Isolation of macrophage population

2.12. Interleukin 1 beta (IL-1b ) release in response to Con A and NPY IL-1b release was measured in the supernatants of macrophage cultures from young, adult, mature and old mice and the procedure to obtain the supernatants was the same used for the TNFa determination that has been explained before. The only difference was the addition of the mitogen Con A Ž1 mgrml. instead of LPS. The IL-1b production was quantified in the supernatants using an R & D System ŽMinneapolis, USA. immunoassay with recombinant mouse IL-1b, a minimum detectable dose of

In some experiments the phagocytosis assay was performed with isolated macrophages from adults and old mice. Briefly, aliquots of peritoneal suspension, previously adjusted at 5 = 10 5 macrophagesrml of Hank’s solution, were dispensed in MIF plates and incubated for 45 min with the addition of trypsin Ž0.5%. in order to remove non-macrophage cells and debris. Then, adhered macrophages were gently washed and incubated with RPMI 1640 medium for up to 24 h. After that, the phagocytosis assay was carried out following the method above described. 2.14. Statistical analysis All values are expressed as the mean " S.D. of the number of experiments, performed in duplicate, which are indicated in the corresponding tables and figures. The data were statistically evaluated by the Mann–Whitney test for non-parametric data, as well as by the Wilcoxon’s test for paired comparisons, with a minimum significance level of p - 0.05.

3. Results The effect of NPY on the adherence capacity of peritoneal macrophages from young, adult, mature and old

Fig. 1. Adherence indexes of murine peritoneal macrophages in the presence of NPY Žfrom 10y1 3 to 10y7 M. at 10 min of incubation time. The results represent the mean " S.D. of eight values corresponding to eight animals, each value being the mean of duplicate assays. The adherence index in young, adult, mature and old with GRP Ž10y1 0 M. were: 39 " 3, 55 " 3 ) ) , 71 " 8 ) ) ) and 60 " 5, respectively; with Ionophore Ž10y7 M. were 48 " 6 ) , 59 " 4 ) ) , 65 " 6 ) ) and 74 " 8 ) ) ; and with fMLP Ž10y8 M. were: 36 " 5, 28 " 6 ) ) ) , 35 " 7 ) ) and 41 " 6 ) ) . In adult age, the assay was performed with lower and higher concentrations of Ionophore Ž10y8 M: 46 " 4, and 10y6 M: 52 " 6. and fMLP Ž10y9 M: 53 " 7, and 10y7 M: 51 " 6.. ) p - 0.05; ) ) p - 0.01; ) ) ) p - 0.001 compared to their respective controls. yy p - 0.01 with respect to young value; a p - 0.05; aa p - 0.01 with respect to adult value; mm p - 0.01 with respect to mature value.

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160

Table 1 Adherence indexes of murine peritoneal macrophages at 20 and 30 min of incubation with NPY 10y1 1 M Young

Adult

Mature

Old

20 min Control NPY

55"5ooo,mm 68"6 )

59"5ooo 76"5 ) ) )

67"8 a,ooo 83"6 ) ) )

84"9 85"3

30 min Control NPY

63"7 a,ooo,mm 75"5 )

70"7 ooo 85"6 ) ) )

72"6 ooo 85"7 ) )

87"5 84"5

Each value is the mean"S.D. of eight experiments corresponding to eight animals, each value being the mean of duplicate assays. ) p- 0.05 With respect to control values. )) p- 0.01 With respect to control values. ))) p- 0.001 With respect to control values. a p- 0.05 With respect to adult values. ooo p- 0.001 With respect to old values. mm p- 0.01 With respect to mature values.

mice after 10 min of incubation is shown in Fig. 1. The controls without neuropeptide showed increasing values of adherence with aging. NPY stimulated significantly the adherence capacity of macrophages in mature and old ages at concentrations between 10y1 3 and 10y7 M, with 10y1 2 , 10y1 1 and 10y8 M as the most effective concentrations used. In young and adult animals, NPY did not affect the adherence capacity of peritoneal macrophages. In order to assess the results obtained with NPY on adherence, positive ŽGRP and ionophore A23187. and negative controls

ŽFMLP. were included. One of the positive control, GRP at 10y1 0 M, stimulated the adherence capacity in adult and mature ages whereas it did not show any significant differences with respect to controls in young and old mice. Ionophore, the other positive control, was used at final concentrations ranging from 10y8 to 10y6 M, with 10y7 M being the only concentration with a significant effect at all ages studied. FMLP, the negative control in adult age, was used at concentrations ranging from 10y9 to 10y7 M, with 10y8 M being the concentration that caused a significant diminution of the adherence index in macrophages from adult, mature and old mice. The changes with aging in the effects of 10y1 1 M NPY, which was the most effective concentration for adherence at 10 min of incubation, have been also studied at 20 and 30 min ŽTable 1.. Again, in controls, the adherence indexes increased with aging. NPY stimulated macrophage adherence capacity at all ages with exception of the old age. Regarding the specificity of the neuropeptide action on adherence, a C-terminal fragment of NPY, i.e: NPY Ž18–36., was used in adults at 20 min of incubation with the 10y1 1 M concentration of NPY. The results were the following: 58 " 6 for the NPY and the antagonist at 10y1 1 M concentration and 57 " 9 for the antagonist alone. These values did not differ significantly from the control Ž59 " 5., thus, confirming the specificity of NPY effects. The effects of NPY on the chemotaxis of peritoneal macrophages at the different ages studied are shown in Fig. 2. Regarding the controls without neuropeptide, a high

Fig. 2. Chemotaxis indexes of murine peritoneal macrophages incubated with NPY Žfrom 10y1 3 to 10y7 M.. The results represent the mean " S.D. of eight values corresponding to eight animals, each value being the mean of duplicate assays. The chemotaxis indexes in young, adult, mature and old with GRP Ž10y1 0 M. were: 365 " 40 ) ) , 1131 " 191) ) ) , 1442 " 192 ) ) and 1348 " 196, respectively; with CCK-8s Ž10y8 M. were 398 " 49 ) ) , 371 " 17 ) ) ) , 894 " 63 ) ) and 1095 " 53 ) ) ; with Ionophore Ž10y7 M. were: 590 " 49 ) ) , 757 " 39 ) ) ) , 2033 " 197 ) ) ) and 2759 " 392 ) ) ) ; and with Retinal Ž2 = 10y5 M. were: 237 " 38 ) ) ) , 414 " 31) , 860 " 63 ) ) , 746 " 50 ) ) ) . In adult age, the assay was performed with lower and higher concentrations of Ionophore Ž10y8 M: 523 " 42 ) , and 10y6 M: 488 " 53. and Retinal Ž2 = 10y6 M: 472 " 28, and 2 = 10y4 M: 485 " 42.. ) p - 0.05; )) p - 0.01 and ) ) ) p - 0.001 compared to their respective controls. yyy p - 0.001 with respect to young value; a p - 0.05; aaa p - 0.001 with respect to adult value; mm m p - 0.001 with respect to mature value.

M. De la Fuente et al.r Journal of Neuroimmunology 116 (2001) 156–167

significant difference was found between mature and old ages, which showed greater indexes of chemotaxis, with respect to young and adults. NPY significantly stimulated the chemotaxis capacity of macrophages in adult age at concentrations ranging from 10y1 3 to 10y8 M, 10y1 2 and 10y1 1 M being the most efficient concentrations. On the contrary, NPY exerted an inhibitory effect on this activity at the other ages studied. NPY decreased the chemotaxis in young animals at all the concentrations used, with the maximum effect ranging from 10y1 1 to 10y8 M. In mature mice, the minimum value with respect to their control was obtained with 10y1 0 M NPY and in old age the highest inhibitory effects were found with 10y1 1 and 10y1 0 M concentrations. The effect of GRP at 10y1 0 M, which is a positive control for adult murine macrophage chemotaxis, caused an increase of this function in young, adult and mature mice, whereas no significant differences with respect to the control were found in old mice. The ionophore increased the chemotaxis capacity of adult mice at 10y8 and 10y7 M, being the highest effect at 10y7 M concentration, which showed a stimulatory effect on chemotaxis capacity at all ages studied. With respect to the negative controls used, the 10y8 M concentration of CCK-8s caused a diminution of chemotaxis in all age groups. In relation to retinal, used at 2 = 10y6 , 2 = 10y5 and 2 = 10y4 M, only the 2 = 10y5 concentration produced a significant decrease of the chemotaxis in adult age. In the other ages, this concentration also acted as an inhibitor of chemotaxis with the highest significance in young and old mice. The data corresponding to the specificity assays for this activity were: 471 " 67 for the NPY 18–36 and NPY Ž10y1 1 M., and 444 " 57 for the NPY 18–36 alone. Again, as occurred in the adherence assay, the NPY effects disappeared when it was added together with this C-terminal fragment. The results obtained for the phagocytic capacity of latex beads are shown in Fig. 3. The control values without neuropeptide were significantly higher in young and old animals with respect to adult and mature mice. NPY increased significantly the phagocytic function of murine peritoneal macrophages from adult and mature animals; in adult mice at concentrations from 10y1 2 to 10y8 M and in mature ones from 10y1 1 to 10y8 M. In young animals, NPY did not show any differences with respect to control values: 482 " 34 Ždata not shown.. In old age, NPY decreased the phagocytic function at concentrations from 10y1 2 to 10y8 M, showing the highest effect at 10y1 1 M. Controls used on the phagocytosis assay such as GRP at 10y1 0 M exerted an stimulant effect in adult mice and in mature mice, whereas no significant effect were found in young and old animals. Ionophore A23187 was also a stimulator of this activity in adult, mature and old ages. CCK-8s at 10y8 M, a negative control, decreased the phagocytic capacity at all ages studied, with its maximum inhibitory effect in old age. EGTA was used as negative control in adult age from 10y4 to 10y2 M, with all concentrations studied being inhibitory of the phagocytic

161

Fig. 3. Number of latex beads phagocytized by 100 macrophages in the presence of NPY Žfrom 10y1 3 to 10y7 M.. Each value is the mean"S.D. of eight experiments corresponding to eight animals, each value being the mean of duplicate assays. The number of latex beads in young, adult, mature and old with GRP Ž10y1 0 M. were: 472"42, 274"22 ) ) , 252"14 ) and 373"38, respectively; with CCK-8s Ž10y8 M. were 435"20 ) ) , 181"27 ) , 170"28 ) ) and 354"27 ) ) ) ; with Ionophore Ž10y7 M. were: 525"46, 371"35 ) ) ) , 313"52 ) ) and 451"25 ) ; and with EGTA Ž10y3 M. were: 406"27 ) ) ) , 143"10 ) ) ) , 200"13 ) ) , 302"28 ) ) ) . In adult age, the assay was performed with lower and higher concentrations of Ionophore Ž10y8 M: 266"33 ) ) , and 10y6 M: 235"28. and EGTA Ž10y4 M: 178"18 ) ) , and 10y2 M: 171"28 ) ) .. ) p- 0.05; ) ) p- 0.01; ) ) ) p- 0.001 compared to their respective controls. yy p- 0.01; yyy p- 0.001 with respect to young. ooo p- 0.001 with respect to old value.

assay with a maximum value at 10y3 M. This concentration was used in the other ages and the same effect was found. Regarding the specificity of the neuropeptide action, two antagonists were used in adults, at concentrations at which the NPY showed the most significant effects, one of them, D-tyr 27,36 D-thr 32 –NPY Ž27–36., was discarded

M. De la Fuente et al.r Journal of Neuroimmunology 116 (2001) 156–167

162

Table 2 Effect of NPY and different controls, GRP, CCK-8s, PMA and Retinal, at the concentration indicated, on superoxide anion production Žnmolr10 6 cells. by murine peritoneal macrophages

Young Adult Mature Old

Control

NPY Ž10y1 0 M.

GRP Ž10y10 M.

CCK-8s Ž10y8 M.

PMA Ž50 ngrml.

Retinal Ž2 = 10y5 M.

35 " 4 aaa,mmm 48 " 3 51 " 2 a 38 " 5aa,mmm

38 " 5 53 " 5 ) 54 " 3 ) 42 " 3

46 " 4 ) ) 59 " 5 ) ) ) 56 " 3 ) ) 42 " 4

35 " 3 39 " 5 ) ) 47 " 6 31 " 4 )

48 " 5 ) ) ) 61 " 7 ) ) 57 " 3 ) ) 50 " 5 ) )

35 " 3 44 " 3 ) 41 " 6 ) ) 43 " 4

Each value is the mean " S.D. of eight experiments corresponding to eight animals, each value being the mean of duplicate assays. ) p - 0.05 With respect to the corresponding control values. )) p - 0.01 With respect to the corresponding control values. ))) p - 0.001 With respect to the corresponding control values. a p - 0.05 With respect to adult value. aa p - 0.01 With respect to adult value. aaa p - 0.001 With respect to adult value. mm m p - 0.001 With respect mature value.

since it showed significant effects by itself on the phagocytosis assays. With this NPY fragment Ž10y1 0 M. alone the number of latex beads ingested by macrophages was 294 " 29, and NPY fragment together with NPY Ž10y1 0 M. was 261 " 26, with no statistically significant differences with respect to the NPY Ž10y1 0 M. Ž288 " 21.. The other C-terminal fragment, NPY Ž18–36., inhibited the effect produced by NPY on the phagocytosis of adult mice, showing a value of 236 " 26 for the NPY and the antagonist Ž10y1 0 M. and of 197 " 20 for the antagonist alone, and for this reason this C-terminal fragment was used in the other assays of the phagocytic process Žadherence, chemotaxis and superoxide anion production.. Regarding superoxide anion production in non-stimulated samples Žwithout ingestion of material., NPY did not exert any significant effect Žin controls the results were: 13 " 3, young; 12 " 3, adult; 16 " 2, mature; 15 " 3, old.. Table 2 shows the superoxide anion production by macrophages stimulated with latex beads as ingestion material and incubated in the presence of the NPY concentration with significant effect Ž10y1 0 M. and the different control compounds. With respect to control values, without neuropeptide, adult and mature animals showed higher production of superoxide anion than young and old mice. The NPY produced a slight but significant increase in

superoxide production, in comparison to the values obtained in the controls, in adult and mature mice, while no differences were found in young and old animals. When macrophages were incubated with PMA Ž50 ngrml., as a positive control, the superoxide anion production was significantly higher than that of NPY in all groups studied. GRP Ž10y1 0 M. stimulated superoxide production at young, mature and adult ages. On the contrary, CCK-8s inhibited the superoxide production in adult mice as well as in old animals, whereas no differences with respect to the control were found in young and mature age. Retinal was also an inhibitor of this assay, but only in adult and mature groups. No change with respect to control was shown when the NPY 18–36 was added together with NPY Ž10y1 0 M. Ž53 " 5.. The value obtained for the antagonist alone was 50 " 4. The capacity of murine peritoneal macrophages for TNFa release in adult and old mice in response to LPS Ž10 mgrml. alone or with NPY Žat concentrations between 10y1 2 and 10y8 M. was also studied. The TNFa release, in response to LPS, in the culture medium of the cells from old mice reached higher Ž p - 0.01. levels Ž1200 " 63 pgrml. than in adult animals Ž626 " 48 pgrml.. When macrophages from animals of both ages were incubated with LPS and NPY, all concentrations used of this neu-

Table 3 IL-1b Žpgrml. release by murine peritoneal macrophages stimulated with Con A Ž1 mgrml. or Con A plus NPY at the different concentrations indicated

Young Adult Mature Old

Control

Con A

NPY Ž10y1 2 M.

NPY Ž10y11 M.

NPY Ž10y10 M.

NPY Ž10y9 M.

NPY Ž10y8 M.

78 " 8 oo 86 " 17 oo 93 " 15 oo 162 " 29

84 " 10 oo 115 " 10 oo,) 121 " 17 oo,) 192 " 31

68 " 5a,) 123 " 12 ) ) 116 " 14 ) 150 " 31

68 " 5a,) 129 " 10 a,) ) 98 " 11a 145 " 25a

77 " 9 119 " 15 ) ) 129 " 12 ) ) 159 " 25

80 " 7 21 " 14 ) ) 118 " 17 ) 149 " 36

85 " 13 116 " 11) ) 114 " 11 181 " 30

The results are the mean " S.D. of five values corresponding to five animals, each value being the mean of duplicate assays. ) p - 0.05 With respect to control values. )) p - 0.01 With respect to control values. a p - 0.05 With respect to Con A values. oo p - 0.01 With respect to the corresponding value in old.

M. De la Fuente et al.r Journal of Neuroimmunology 116 (2001) 156–167

ropeptide exerted similar inhibitory effect on the release of TNFa. For example, the values obtained for 10y1 0 M of NPY were 509 " 27 pgrml for adult Ž p - 0.01 with respect to LPS value. and 961 " 72 for old age Ž p - 0.05 with respect to LPS value.. The IL-1b levels at the four ages studied in response to NPY and Con A mitogen Ž1 mgrml. are shown in Table 3. In controls and after Con A stimulation, the release of IL-1b was significantly higher in old mice with respect to the other ages studied. The stimulation of IL-1b release by Con A was significantly increased in the presence of NPY Ž10y1 1 M. in adult mice, while it was decreased for the remaining ages studied. In order to investigate the intracellular pathways implicated in the NPY effects, different activators and inhibitors

163

of adenylate cyclase and protein kinase C were used. The different effects of these activators and inhibitors on the phagocytic activity of macrophages from adult and old mice are represented in Fig. 4. In adult age, a well-known stimulator of adenylate cyclase, i.e.: forskoline, inhibited the phagocytosis, but when it was added together with NPY, the inhibition disappeared. Adenosine, a known inhibitor of adenylate cyclase, by itself and together with NPY stimulated the phagocytic process. With respect to the PKC pathway, macrophages incubated with PMA, a specific activator of this kinase, showed a stimulation of phagocytosis and the same occurred when NPY was added together with PMA. Retinal inhibited significantly the phagocytic activity, and this effect disappeared when NPY was added. In old mice forskoline showed the same effect

Fig. 4. Phagocytosis of adult and old mice in the presence of NPY Ž10y1 2 and 10y8 M., forskoline ŽF., adenosine ŽA., phorbol myristate acetate ŽPMA. and retinal ŽR.. The results represent the mean " S.D. of eight values corresponding to eight animals, each value being the mean of duplicate assays. ) p - 0.05; ) ) p - 0.01 with respect to corresponding control values.

M. De la Fuente et al.r Journal of Neuroimmunology 116 (2001) 156–167

164

Table 4 cAMP levels Žpmolr10 8 cells. in isolated murine peritoneal macrophages from adult and old mice incubated for 30 or 60 s with Forskoline ŽFK., Adenosine ŽA., GRP, CCK-8s or NPY at the concentrations indicated Control

FK Ž10y5 M.

A Ž10y7 M.

GRP Ž10y10 M.

CCK-8s Ž10y10 M.

NPY Ž10y12 M.

NPY Ž10y10 M.

NPY Ž10y8 M.

Adult 30 s 60 s

43 " 9 55 " 5

44 " 7 54 " 6

29 " 5 ) 51 " 8

32 " 3 ) 51 " 8

48 " 5 81 " 9 ) )

53 " 3 ) 59 " 5

59 " 7 ) 60 " 7

44 " 6 52 " 8

Old 30 s 60 s

51 " 7 50 " 7

54 " 6 45 " 6

47 " 10 54 " 6

38 " 5 ) ) 42 " 9

57 " 7 47 " 8

45 " 7 53 " 8

46 " 9 49 " 6

40 " 5 ) 47 " 8

The results are the mean " S.D. of five values corresponding to five animals, each value being the mean of duplicate assays. ) p - 0,05 With respect to control values. )) p - 0,01 With respect to control values.

than in adults i.e.: a decrease of the ingestion, but with the addition of NPY an even higher decrease of phagocytosis was observed with respect to forskoline or NPY alone. No effect was shown with adenosine with respect to controls and, with the addition of NPY, the same decrease of ingestion than with the NPY alone was observed. No significant differences appeared when PMA was used and a smaller ingestion, as compared to control, was observed when cells were incubated with PMA and NPY. Retinal inhibited the phagocytic process and this effect was more significant when NPY was added together with it. In addition to the study of the mechanisms implicated in the NPY action, the intracellular levels of cAMP were measured. The results ŽTable 4. show an increase of cAMP in the macrophages from adults and a decrease in cells

from old mice in response to NPY. Since these results did not agree with those obtained in phagocytosis, we performed the phagocytosis assay with a purified population of macrophages, without peritoneal lymphocytes. In these isolated populations, the number of latex beads phagocyted ŽFig. 5. decrease or increase in response to NPY in adult and old mice, respectively, in agreement with the cAMP measurement previously performed on the same purified population.

4. Discussion Recently, changes with aging in the effects of several neuropeptides on immune functions such as lymphocyte

Fig. 5. Number of latex beads phagocytized by 100 cells in total peritoneal suspension Žwhite bars. and in purified peritoneal macrophages Žhatched bars. in the presence of NPY Ž10y1 2 and 10y8 M.. Each value is the mean " S.D. of eight experiments corresponding to eight animals, each value being the mean of duplicate assays. ) p - 0.05, ) ) p - 0.01 with respect to the controls; vv p - 0.01 with respect to the total population.

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adherence and chemotaxis ŽMedina et al., 2000b., lymphoproliferative capacity ŽMedina et al., 2000a. and NK activity ŽMedina et al., 1998. have been reported. Some neuropeptides such as GRP ŽDe la Fuente et al., 1991., CCK-8s ŽDe la Fuente et al., 1995a., VIP ŽDe la Fuente et al., 1993b. or NPY itself ŽDe la Fuente et al., 1993a. modulate phagocyte functions, but this modulation may change with aging ŽDe la Fuente et al., 2000.. In the present work, the variations with aging in the modulation exerted by NPY on different macrophage functions have been found. Moreover, the effects occur at the same concentrations at which they have been shown to produce the effect on the other immune cells, and these effects are carried out through specific receptors and intracellular pathways. The first step in phagocytosis involves the adherence of phagocytic cells to tissue substrate before migration of these cells to the site of inflammation. With aging, an increase in the adherence of murine macrophages has been reported ŽMcArthur, 1998.. NPY was shown to increase the adhesiveness of leukocytes to endothelial cells suggesting a role of this peptide in this cellular process ŽSung et al., 1991.. In the present work, NPY increased the adherence in cells from mature and old mice. The norepinephrine, a neurotransmitter which has been shown to release by noradrenergic neurons along with NPY, also increase adherence of peritoneal macrophages from BALBrc mice at physiological concentrations of 10y1 2 M at all the same ages studied in the present work ŽOrtega et al., 2000a.. A stimulation of adherence should be interpreted with caution since it does not necessarily means a favorable action on immune function, unless a stimulation of the consequent steps of the phagocytic process occurs. Thus, some neuropeptides, like cholecystokinin or gastrin, inhibit phagocyte functions and stimulate the adherence of these immune cells ŽDe la Fuente et al., 1995a.. An adequate chemotaxis capacity allows the phagocyte to reach and accumulate at the inflammation sites and is essential for ensuing phagocytosis of foreign or damaged material. In the present study we have found a greater chemotaxis index in macrophages from aged mice, in agreement with previous work ŽOrtega et al., 2000a, b.. A possible explanation for this significantly augmented chemotaxis indexes found in old with respect to adult mice could be a release of chemokines induced by the higher release of proinflammatory cytokines i.e.: TNFa and IL-1b in old animals ŽSegal et al., 1997; Rink et al., 1998.. The chemotaxis of macrophages from adult mice was stimulated by NPY at all the concentrations studied. By contrast, the chemotaxis of macrophages from young, mature and old animals was decreased by NPY. An interesting possibility is that the decreasing effect of NPY on mature and old mice brings the chemotaxis indexes closer to the adult values. In fact, norepinephrine showed similar results, a decline of this capacity in mature and old mice at high concentrations Ž10y5 to 10y7 M. ŽOrtega et al., 2000a..

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Previously, we had observed a similar behaviour of NPY on peritoneal lymphocyte chemotaxis of adult and old mice ŽMedina et al., 2000b.. A similar effect to that of NPY on chemotaxis was observed for phagocytosis. Thus, NPY increased this activity in macrophages from adult confirming previous results ŽDe la Fuente et al., 1993a. and from mature mice while it decreased the phagocytosis of old mice, which was almost two-fold higher than in adult animals. The great phagocytosis index observed in the present study in cells from old mice has been previously found in other work and related with the oxidative situation of old animals ŽOrtega et al., 2000b.. NPY seems again to have a modulatory role on the changes in this immune function with aging, keeping it on presumably optimal values. In the presence of a phagocytic stimulus, macrophages initiate what is known as the respiratory burst, in which NADPH oxidase is activated catalyzing a reaction that . produces superoxide anion ŽOy 2 , which is a precursor of the active microbicidal oxidants ŽSegal and Abo, 1993.. The production of superoxide anion by macrophages in response to the phagocytic stimulus was greater in adult and mature mice than in the young and old, and NPY increased this production of oxygen radicals in adult and mature animals but not in the young and old. This fact could reveal other aspects of the immunomodulatory role of NPY, since the production of free radicals, which are necessary for the phagocyte function, is only increased in cells from adult and mature mice which have adequate antioxidant levels for neutralizing the possible nocive effect of superoxide anion, while cells from old animals show reduced endogenous antioxidants defenses ŽDe la Fuente et al., 1995b.. Since the specificity of the effects observed was evaluated with two C-terminal fragments of NPY, i.e., NPY Ž18–36., previously described with agonist andror antagonist properties for Y1 , Y2 , and Y3 receptors ŽBalasubramaniam and Sheriff, 1990; Levite et al., 1998. and Dtyr 27,36 D thr 32 –NPY Ž27–36. which acts as an antagonist at one or more subtypes of the NPY receptor in the rat brain ŽY1 and Y2 receptors. ŽMyers et al., 1995., the results obtained in the present work suggest that the effects exerted by NPY on the phagocytic process of murine peritoneal macrophages are at least, mediated through any kind of those receptors. Moreover, we have also shown that D-tyr 27,36 D-thr 32 –NPY Ž27–36. behaves as an agonist for peritoneal macrophages exerting qualitatively similar effects to those produced by NPY. In addition to the phagocytic process, the release of cytokines by macrophages is also modulated by NPY. The release of TNFa in response to LPS was higher in old than in adult mice as it had been already reported ŽSegal et al., 1997; Rink et al., 1998.. NPY decreased this production of TNFa , which can be interpreted as a positive modulation of NPY because TNFa is involved in many diseases and inflammatory processes ŽKimble, 1997.. Re-

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garding the release of IL-1b, it was higher in old than in adult animals, which is in agreement with other work ŽHernanz et al., 1996; Segal et al., 1997., and NPY acted stimulating the release of this cytokine in adults while inhibiting it in old mice. Again, NPY seems to play an homeostatic role, keeping the amount of this cytokine at optimal levels, taking into account that this is a proinflammatory cytokine ŽRink et al., 1998. but at the same time necessary for lymphoproliferation and for the success of other immunological functions ŽDurum and Oppenheim, 1989.. The phagocytosis assay was chosen for studying the mechanisms of action of NPY. It has been reported that high levels of cAMP and low levels of PKC are correlated with a decrease in several phagocytic functions such as chemotaxis and phagocytosis. On the contrary, low levels of cAMP and high levels of PKC, accompany an increased of these functions ŽDe la Fuente et al., 1991, 1993a; De la Fuente, 1999.. In this work, confirming results have been obtained i.e., in the peritoneal suspension from adult mice, NPY inhibits adenylate cyclase, stimulates PKC and increases phagocytosis. However, phagocytosis is inhibited by NPY and at the same time cAMP pathway is activated and PKC is decreased in old mice. Moreover, NPY increases chemotaxis in adult mice, while it decreases them in old mice. Thus, intracellular mechanisms seems to be reverted in aging. The action of NPY on phagocytosis of macrophages depend on the presence of lymphocytes as has been shown in the present work. In fact in isolated macrophages from adult mice NPY increases cAMP levels and decreases phagocytosis, whereas in these isolated cells from old mice NPY decreases cAMP levels and increases phagocytosis. However, these two cells are always together in vivo in the peritoneum and in other immune locations, and the changes in the NPY effects with aging appear, although in an opposite way, in both isolated and non isolated macrophages. Therefore, the changes with age in the effect on NPY could be linked to changes in the collaboration between macrophages and lymphocytes. In the context of the neuro-immune communication, NPY seems to act as a phagocytic cell modulator, not only in adult age but also at all ages studied in this work Ži.e.: young, adult, mature and old.. When a stimulation of the sympathetic nervous system and the consequent NPY release occurs, the response of phagocytes to this nervous signal depend on the animal age studied. In conclusion NPY could exert a regulatory role maintaining the phagocytic functions at a physiologically optimal level.

Acknowledgements We thank Dr. J. Miquel for the critical reading of the manuscript. This work was supported by Grants from FISss ŽNo 99r0815..

References Bagasra, O., Howeedy, A., Kajdacsy-Balla, A., 1998. Macrophage function in chronic experimental alcoholism. Modulation of surface receptors and phagocytosis. Immunology 65, 405–409. Balasubramaniam, A.A., Sheriff, S., 1990. Neuropeptide Y Ž18–36. is a competitive antagonist of neuropeptide Y in rat cardiac ventricular membranes. J. Biol. Chem. 265, 14724–14727. Besedovsky, H., Del Rey, A., 1996. Immune-neuro-endocrine interactions: facts and hypotheses. Endocr. Rev. 17, 67–102. Boyden, S.V., 1962. The chemotaxis effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115, 453–466. Daniels, A.J., Lazarowski, E.R., Matthews, J.E., Lapetina, E.G., 1989. Neuropeptide Y mobilizes intracellular Ca2q and increases inositol phosphate production in human erytroleukemia cells. Biochem. Biophys. Res. Commun. 165, 1138–1144. De la Fuente, M., 1999. Modulation of immune function by neuropeptides. Curr. Trends Immunol. 2, 111–122. De la Fuente, M., Del Rıo, ´ M., Ferrandez, M.D., Hernanz, A., 1991. Modulation of phagocytic function in murine peritoneal macrophages by bombesin, gastrin-releasing peptide and neuromedin C. Immunology 73, 205–211. De la Fuente, M., Bernaez, I., Del Rıo, ´ M., Hernanz, A., 1993a. Stimulation of murine peritoneal macrophage functions by neuropeptide Y and peptide YY. Involvement of protein kinase C. Immunology 80, 259–265. De la Fuente, M., Delgado, M., Del Rıo, C., Hernanz, A., ´ M., Martınez, ´ Gomariz, R.P., 1993b. Stimulation by vasoactive intestinal peptide ŽVIP. of phagocytic function in rat macrophages. Protein kinase C involvement. Regul. Pept. 48, 345–353. De la Fuente, M., Campos, M., Del Rıo, ´ M., Hernanz, A., 1995a. Inhibition of murine peritoneal macrophage functions by sulfated cholecystokinin octapeptide. Regul. Pept. 55, 47–56. De la Fuente, M., Hernanz, A., Collazos, M.E., Barriga, C., Ortega, E., 1995b. Effects of physical exercises and ageing on ascorbic acid and superoxide anion levels in peritoneal macrophages from mice and guinea pigs. J. Comp. Physiol., B 165, 315–319. De la Fuente, M., Medina, S., Del Rıo, M.D., Hernanz, ´ M., Ferrandez, ´ A., 2000. Effect of aging on the modulation of macrophage functions by neuropeptides. Life Sci. 67, 2125–2135. Delgado, M., Garrido, E., De la Fuente, M., Gomariz, R.P., 1996. Pituitary adenylate cyclase-activating polypeptide ŽPACAP38. stimulates rat peritoneal macrophage functions. Peptides 17, 1097–1105. Durum, S.K., Oppenheim, J.J., 1989. Macrophage-derived mediators: interleukin 1, tumor necrosis factor, interleukin 6, interferon and related cytokines. In: Paul, W.E. ŽEd.., Fundamental Immunology. Raven Press, New York, pp. 639–662. Fabris, N., 1991. Neuroendocrine-immune interactions: a theoretical approach to ageing. Arch. Gerontol. Geriatr. 12, 219–230. Friedman, E.M., Irwin, M.R., 1997. Modulation of immune cell function by the autonomic nervous system. Pharmacol. Ther. 74, 27–38. Garrido, E., Delgado, M., Martınez, C., Gomariz, R.P., De la Fuente, M., ´ 1996. Pituitary adenylate cyclase-activating polypeptide ŽPACAP38. modulates lymphocyte and macrophage functions: stimulation of adherence and opposite effect on mobility. Neuropeptides 30, 583–595. Gehlert, D.R., Gackenheimer, S.L., 1996. Unexpected high density of neuropeptide Y Y1 receptors in the guinea pig spleen. Peptides 17, 1345–1348. Ginaldi, L., De Martinis, M., D’Ostilio, A., Marini, L., Loreto, M.F., Quaglino, D., 1999. Immunological changes in the elderly. Aging Clin. Exp. Res. 11, 281–286. Goya, R.G., Brown, O.A., Bolognani, F., 1999. The thymus–pituitary axis and its changes during aging. Neuroimmunomodulation 6, 137– 142. Hernanz, A., Tato, E., De la Fuente, M., de Miguel, E., Arnalich, F., 1996. Differential effects of gastrin-releasing peptide, neuropeptide Y, somatostatin and vasoactive intestinal peptide on interleukin-1b, inter-

M. De la Fuente et al.r Journal of Neuroimmunology 116 (2001) 156–167 leukin-6 and tumor necrosis factor-a production by whole blood cells from healthy young and old subjects. J. Neuroimmunol. 71, 25–30. Johansson, O., Sandberg, G., 1989. Effect of the neuropeptides b-MSH, neurotensin, NPY, PHI, somatostatin and substance P on proliferation of lymphocytes in vitro. Acta Physiol. Scand. 137, 107–111. Kimble, R.B., 1997. Alcohol, cytokines and estrogens in the control of bone remodelling. Alcoholism. Clin. Exp. Res. 21, 385–391. Levite, M., Cahalon, L., Hershkoviz, R., Steinman, L., Lider, O., 1998. Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin. J. Immunol. 160, 993–1100. McArthur, W.P., 1998. Effect of aging of immunocompetent and inflammatory cells. Periodontology 2000 16, 53–79. Medina, S., Del Rıo, ´ M., Ferrandez, M.D., Hernanz, A., De la Fuente, M., 1998. Changes with age in the modulation of natural killer activity of murine leukocytes by gastrin-releasing peptide, NPY and CCK-8s. Neuropeptides 32, 549–555. Medina, S., Del Rıo, ´ M., Hernanz, A., De la Fuente, M., 2000a. Age-related changes in the neuropeptide Y effects on murine lymphoproliferation and interleukin-2 production. Peptides 21, 1403–1409. Medina, S., Del Rıo, ´ M., Hernanz, A., De la Fuente, M., 2000b. The NPY effects on murine leukocyte adherence and chemotaxis change with age. Adherent cell implication. Regul. Pept. 95, 35–45. Myers, R.D., Wooden, M.H., Ames, C.D., Nyce, J.W., 1995. Anorexic action of a new potential neuropeptide Y antagonist ŽD-Tyr Ž27,36., D-thr Ž32.. –NPY Ž27–36. infused into the hypothalamus of the rat. Brain. Res. Bull. 37, 237–245. Noga, S.J., Norman, S.J., Weiner, R.S., 1984. Methods in laboratory investigation: isolation of guinea pigs monocytes and Kurloff cells. Characterization of monocyte subsets by morphologic, cytochemistry and adherence. Lab. Invest. 51, 244–249. Ortega, E., Barriga, C., De la Fuente, M., 1993. Aging and the non-specific immune response. Facts Res. Gerontol. 7, 23–29. Ortega, E., Garcıa, ´ J.J., De la Fuente, M., 2000a. Modulation of adherence and chemotaxis of macrophages by norepinephrine. Influence of ageing. Mol. Cell. Biochem. 203, 113–117.

167

Ortega, E., Garcıa, ´ J.J., De la Fuente, M., 2000b. Aging modulates some aspects of the non-specific immune response of murine macrophages and lymphocytes. Exp. Physiol. 85, 519–525. Pawelec, G., 1999. Immunosenescence: impact in the young as well as the old? Mech. Ageing Dev. 108, 1–7. Rink, L., Cakman, I., Kirchner, H., 1998. Altered cytokine production in the elderly. Mech. Ageing Dev. 102, 199–209. Romano, T.A., Felten, S.Y., Felten, D.L., Olschowka, J.A., 1991. Neuropeptide Y innervation of the rat spleen: another potential immunomodulatory neuropeptide. Brain, Behav., Immun. 5, 116–120. Segal, A.W., Abo, A., 1993. The biochemical basis of the NADPH oxidase of phagocytes. Trends Biochem. Sci. 18, 43–47. Segal, R., Dayan, M., Globerson, A., Habut, B., Shearer, G.M., Mozes, E., 1997. Effect of aging on cytokine production in normal and experimental systemic lupus erythematosus afflicted mice. Mech. Ageing Dev. 96, 47–58. Soder, O., Hellstrom, ¨ ¨ P., 1987. Neuropeptide regulation of human thymocyte, guinea pig T lymphocyte and rat B lymphocyte mitogenesis. Int. Arch. Allergy Appl. Immunol. 84, 205–209. Sung, C.P., Arleth, A.J., Feuersteim, G.Z., 1991. Neuropeptide Y up regulates the adhesiveness of human endothelial cells for leukocytes. Circ. Res. 68, 314–318. Unanue, E.R., 1989. Macrophages, antigen-presenting cells, and the phenomena of antigen handling and presentation. In: Paul, W.E. ŽEd.., Fundamental Immunology. Raven Press, New York, pp. 95–115. Vıctor, V.M., Guayerbas, N., Puerto, M., Medina, S., De la Fuente, M., ´ 2000. Ascorbic acid modulates in vitro the function of macrophages from mice with endotoxic shock. Immunopharmacology 46, 89–101. Westlind-Danielsson, A., Unden, A., Abens, J., Andell, S., Bartfai, T., 1987. Neuropeptide Y receptors and the inhibition of adenylate cyclase in the human frontal and temporal cortex. Neurosci. Lett. 74, 237–242.