Respiratory burst reaction changes with age in rat peritoneal macrophages

Biochimica et Biophysica Acta, 1179 (1993) 247-252 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4889/93 /$06.00

247

BBAMCR 13468

Respiratory burst reaction changes with age in rat peritoneal macrophages Elolsa Alvarez, Consuelo Santa Maria* and Alberto Machado Departamento de Bioqulmica, Bromatolog[a y Toxicolog[a, Facultad de Farmacia. Universidad de Sevilla, C/Prof. Garda Gonzdlez s /n, 41012 Sevilla (Spain) (Received 20 April 1993)

Key words: Macrophage; Respiratory burst; Immune function; Aging; (Rat)

The respiratory burst reaction, estimated as O~- production, has been studied in rat peritoneal macrophages of different age (3, 12 and 24 months). To stimulate NADPH oxidase, the enzyme responsible for the respiratory burst, various stimuli that act in different ways have been used: PMA (phorbol myristate acetate), Con-A (concanavalin A) and N-FMLP (N-formyl-methionylleucyl-phenylalanine). All produced a decrease in response with age, with that from PMA being the greatest. The PMA-induced decrease in the 0 2- production may be related to the inactivation of NADPH-producing enzymes such as glucosc-6-phosphate dehydrogenase and 6-phosphogluconate dchydrogenase that we have found with age. Glutathione reductase, an enzyme that participates in the maintenance of the redox status in the ceil, also showed an age-related decrease. Enzymes that participate in oxygen species scavenging, such as glutathione peroxidase and C u / Z n superoxide dismutase, did not change with age, although an age-related decrease in catalase activity was found.

Introduction The elderly have a higher incidence of mortality due to bacterial infections than do young adults [1], and this change is associated with a number of impaired immune responses [2]. It has long been clear that aging leads to a substantial decline in most T and B-cell functions [3,4]. However, the effects of aging on macrophages, that play a critical role in the immune response, are quite contradictory. Some authors reported no decrease of macrophage function with aging, others a decrease, while still others found an increased functional capacity of the monocyte-macrophage system in old animals [5,6]. Macrophages are the most important phagocytic cells and the most directly involved in the defense against infections. They appear to be involved in antigen presentation, as well as in the secretion of factors which stimulate lymphocytes. Macrophages also have the capacity to kill microbes and tumor cells by generating reactive oxygen species upon interaction with phagocytic stimuli or soluble agents by a metabolic pathway called respiratory burst. Respiratory burst is a coordinate series of metabolic events that lead to the produc-

* Corresponding author.

tion of oxygen free radicals [7]. The superoxide anion is the initial product of the respiratory burst [8]. It is formed by the monoelectronic reduction of 0 2, with N A D P H as the specific electron donor [9]. The active enzyme complex which catalyzes the generation of O2from 0 2 is called N A D P H oxidase; it is composed of both membrane and cytosolic proteins. A continuous supply of N A D P H in phagocytic cells is ensured by the pentose phosphate pathway. The respiratory burst is induced in macrophages by exposure to appropriate stimuli which activate the O2generating enzyme. It is known that the oxidase may be activated besides stimulation of the phagocytes with a wide variety of stimuli which act through binding to receptors other than the Fc and C3h receptors that mediate phagocytoses. These stimuli include: N-formyl-methionyl-leucyl-phenylalanine ( N - F M P L ) [10], phorbol myristate acetate (PMA)[11], concanavalin A (Con-A) [12] and others [13,14]. In order to achieve a better understanding of the behavior of the macrophage in aging, we studied the respiratory reaction, estimated as 0 2- production of rat peritoneal macrophages, in function of age, using several stimuli. We also investigated the effect of age on the activity of some pentose phosphate shunt enzymes and enzymes involved in oxygen species scavenging.

248 Materials and Methods

Animals. Young (3 months), mature (12 months) and old (24 months) female Wistar rats were used. They were maintained on a standard laboratory diet with free access to food and water. Chemicals. Stock solutions of phorbol 12-myristate 13-acetate (PMA, Sigma) were dissolved at 1 m g / m l in dimethylsulfoxide (DMSO, Sigma) and stored at -20°C. Stock solutions of N-formyl-methionyl-leucylphenylalanine (N-FMLP, Sigma) and cytochalasin B (cyto B, Sigma)were prepared in DMSO and stored at -20°C. Solutions of concanavalin A (Con-A, Sigma) were prepared before its use in Krebs-Ringer bicarbonate buffer (KRB (pH 7.4)). Biochemicals were from Boehringer-Mannheim (Mannheim, Germany) or Sigma (St. Louis, MO, USA). Other chemicals were of analytical grade from Merck (Darmstadt, Germany). Preparation of peritoneal macrophages. Peritoneal macrophages were elicited from Wistar rats according to the Tsunawsky and Nathan method [15]. Female Wistar rats, allowed free access to food and water, were injected intraperitoneally 4 days before harvest with 5 ml of 6% sodium caseinate. Animals were killed by decapitation and, immediately, peritoneal cavity was washed with 10 ml of saline solution. Ceils were pelleted by centrifugation, resuspended in KRB and immediatelly used for experiments. Viability, as determined by Trypan-blue exclusion, was always higher than 95%. The recruitment of cells to the peritoneal cavity by the caseinate was similar in all the ages studied ((14.71 + 1.56)- 10 ~ in 3-month-old rats, (17.12 + 1.71). 106 in 12-month-old rats and (15.98 + 1.38). 106 in 24-monthold rats). Determination of 02-. Ceils ((1-1.5). 106 c e i l s / m l in KRBo, with 10 mM glucose) were prewarmed at 37°C with 80 p,M cytochrome c for 5 min prior to the addition of stimulant agents. The reduction of cytochrome c was recorded continuously using a spectrophotometer at 550 nm. 0 2- production was estimated by measuring the superoxide dismutase-inhibitable reduction of cytochrome c at 37°C, as previously described [16], using a molar absorption coefficient of 21.1 mM - l c m t Preparation of peritoneal macrophages for enzyme assays. Thc peritoneal macrophages were obtained using the general method described elsewhere [15] and were then sonicated at 4°C 3-4-times 10 s. Cell disruption was monitored using the Trypan-bluc exclusion method. Centrifugation at 800 x g for 10 rain and the 800 × g supernatant was used to determine enzyme activities. Enzyme assays. 6-Phosphogluconate dehydrogenase was measurcd as described by Levy et al. [17] and

modified by Dror et al. [18]. The reaction mixture contained: 50 mM Tris-HCl (pH 7). 1.5 mM CI, Mg, 0.25 mM NADP, 0.5 mM 6-phosphogluconic acid and rnacrophagcs sample. The reaction was initiated by the adition of 6-phosphogluconic acid and the reduction of NADP was followed spectrophotometrically at 341) nm. Glucose-6-phosphate dehydrogenase was measured as the same method but using glucose 6-phosphate as substrate. Glutathione peroxidase activity was assayed with a coupled enzyme system in which GSSG reduction was coupled to NADPH oxidation by glutathionc reductase [19]. The assay mixture contained phosphate buffer (50 mM potassium phosphate, 1 mM EDTA, 1 mM NaN 3 (pH 7)), 1 mM GSH, 0.2 mM NADPH, 1 U glutathione reductase and macrophage samples. After 5 min of preincubation (20-22°C), the reaction was initiated by the addition 0.1 ml of 0.25 mM HzO 2 (final volume 1.0 ml). Glutathione reductase activity was determined spectrophotometricaily by measuring N A D P H oxidation at 340 nm [20]. The reaction mixture contained, phosphate buffer (20.5 mM potassium phosphate, 26.46 mM dipotassium phosphate, 200 mM KCI, 1 mM EDTA) pH 7, 0.1 mM N A D P H and macrophage samples. After 5 min of preincubation (37°C), the reaction was initiated by the addition of 0.1 ml of 1 mM GSSG. Superoxide dismutase activity was measured using the xanthin-oxidase-cytochrome c method as described by McCard and Fridovich [21]. The final concentrations in the cuvettes were 50 mM potassium phosphate (pH 7.8), 0.1 mM EDTA, 10/zM cytochrome c, 50 # M xanthin, 50 g M or 2 mM cyanide, 1 U catalase. The reaction was initiated by the addition of 1 U xanthinoxidase. The inhibition of xanthin-oxidase was followed spectrophotometrically at 550 nm. Catalase activity was assayed according to the method of Beers and Sizer [22], measuring the decrease in the absorbance at 240 nm at 25°C after the addition of substrate: H20~- final concentration I(X) mM. All spectrophotometric measurements were carried out in a Shimadzu 160 A ultraviolet spectrophotometer with 1.0-ml quartz cuvettes with a light-path of 1.0 cm. All enzymes assays were performed at 25°C, except for glutathione reductase which was assayed at 37°C. Specific activities were expressed as n m o l / m i n per mg protein. Protein concentrations were determined by the method of Lowry, as modified by Markwell et al. [23], with serum bovine albumin as the standard. Statistical methods. Student's t-test was utilized to evaluate differences between means, and the 0.05 level of probability was used as the criterion of significance. All values reported are means _+ S.D. of at least five animals.

249 Results

8 PMA

0 2- production by rat macrophages stimulated with different agents The tumoral promoter PMA has been described as the most potent stimulating agent of respiratory burst [24]. Apparently, PMA binds to and activates a phospholipid-dependent protein kinase C which is then translocated to the membrane where it activates NADPH oxidase [25]. In this process there is no hydrolysis of phosphoinositides using PMA as stimuli. We have found that macrophages display a progressive reduction in their capacity to produce 0 2- which increasing age (Fig. IA). The decrease was from 6.21 _+ 0.4 nmol O2-/min per mg protein in 3-month-old rats to 3.00 + 0.56 nmol O2-/min per mg protein in 24month-old rats. The differences found between the three ages studied were in all cases statistically significant. Synthetic N-formyl-methionyl peptides are active chemotactic agents for polymorphonuclear neutrophils and macrophages, although less so than PMA [26]. N-FMLP induces a GTP-binding protein-linked activation of phosphodiesteric hydrolysis of phosphatidylinositol 4,5-biphosphate with formation of inositolphosphates and Ca 2+ mobilization and protein phosphorylation [27]. When we stimulated macrophages with 1 /xM N-FMLP + cytochalasine B (a drug that increases 0 2- production in macrophages stimulated with the chemotactic peptide and not with other agents) we observed a slight increase (not significant) in 12month-old rats; afterwards the response decreased and the values found in 24-month-old rats were approx. 35% lower than in young animals (Fig. IB). Similarly to the chemotactic peptide, Con-A activates the hydrolysis of phosphatidylinositol 4,5-bisphosphate. However, Con-A differs from N-FMLP in that it bypasses the GTP-binding protein, and its activation is not affected by pertussis toxin and does not produce protein kinase C translocation either [27]. As with the chemotactic peptide, we found the highest values in the middle-aged animals (12 months) although the differences were not significant with respect to young rats (3 months). In aging, Oj- production decreased, showing significant differences with the values found in 12- and 3-month-old rats (Fig. 1C).

Enzyme actiuities The two important enzymes of the pentose phosphate shunt are glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The NADPH is supplied to the respiratory burst via this route. Glucose-6-phosphate dehydrogenase activity of rat macrophages has been investigated in relation to age (Fig. 2A). The activity measured in 24-month-oid rats was approx. 70% lower than in 3-month-old rats. The

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Fig. 1. 0 2- production by peritoneal macrophages induced by various stimulants (PMA, 100 nM; N-FMLP, 1 /xM; Con-A, 50 tzg/ml) in rats of different ages. The results are means + S.D. from at least five separate experiments. Statistical significance: a (difference between 24 months and 3 months), b (difference between 24 months and 12 months), c (difference between 12 months and 3 months). For PMA; a ( P <0.001), b ( P < 0 . 0 1 ) , c ( P < 0 . 0 0 1 ) . For N-FMLP; a ( P < 0.02), b ( P < 0.005). For Con-A: a ( P < 0.01), b ( P < 0. (KI5).

value observed at 12 months was also significantly different from that at 3 months (there was a decrease of 56.6%). Between 12 and 24 months there was a slight but significant decrease (about 30%). The activity of 6-phosphogluconate dehydrogenase in rat peritoneal macrophages was similar (Fig. 2B). The values were 95.36 +_4.12 nmol/min per mg protein, 49.96 _+ 1.07 nmol/min per mg protein and 36.3 _+6.75 nmoi/min per mg protein in rats of 3, 12 and 24 months, respectively. These results show that there is a loss of about 60% of macrophage phosphogluconate dehydrogenase activity with aging. The aging pattern of glutathione reductase in rat peritoneal macrophages is shown in Fig. 3A. Glu-

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Fig. 2. Specific activity of glucose-6-phosphatehosphate dehydrogenase (A) and 6-phosphogluconate dehydrogenase (B) in macrophages of different ages (3, 12 and 24 months). The results arc means_+ S.D. from at least five separate experiments. Statistical significance: a (difference between 24 and 3 months), b (difference between 24 and 12 months), c (difference between 12 and 3 months). For A; a (P < 0.001), b (P < 0.05), c ( P < 0.001). F o r B; a ( P < 0.001). b ( P < 0.003). c ( P < 0.01).

tathione reductase is an important enzyme for the maintenance of glutathione in the reduced form, and possibly for controlling the redox state of N A D P in tissue, if GSSG is available [28]. The enzyme activity decreased from 3 to 12 months (about 40%), afterwards undergoing a slight an non-significant decrease between 12 and 24 months. The specific activities of C u / Z n superoxide dismutase, glutathione peroxidase and catalase, enzymes considered to be specifically involved in the defense of the cell against the partially reduced forms of oxygen, were determined in rat peritoneal macrophages in

function of age (Fig. 3B,C,D). Neither glutathione peroxidase nor C u / Z n superoxide dismutase varied significantly with age. However, catalase activity underwent an age-decrease; the values were 76.14 + 4.63 n m o l / min per mg protein, 57.81 + 7.32 n m o l / m i n per mg protein and 51.81 + 2.23 n m o l / m i n per mg protein in rats of 3, 12 and 24 months, respectively. Discussion At present, the results concerning the effect of aging upon mononuclear phagocytes are inconclusive. Some

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Fig. 3. Specific activity of glutathionc reductase CA), glutathione peroxidase (B), C u / Z n superoxide dismutase (C) and catalase (D) in macrophages of different ages (3, 12 and 24 months). The results are means ± S.D. from at least five ,separate experiments. Statistical significance: a (difference between 24 and 3 months), b (difference between 24 and 12 months), c (difference between 12 and 3 months). For A; a ( P < 0.001), c ( P < 0.001). F o r D; a ( t ' < 0.001). c ( P < 0. (1{)2).

251 authors indicate that several macrophage-dependent host defense mechanisms are decreased [29,30], apparently unaffected or even enhanced with age [6]. Macrophages play an important role in the immune response because they are involved in antigen presentation, in the secretion of factors which stimulate lymphocytes, and killing microbes and tumor cells by generating reactive oxygen species such as anion superoxide (O2). We have investigated the effect on 0 2- production in rat peritoneal macrophages of different ages of agents that act in different ways. The stimuli used were PMA, N-FMLP and Con-A. PMA activates a phospholipid-dependent protein kinase C producing N A D P H oxidase activation. The N-FMLP induces a GTP-binding protein-linked activation of phosphodiesteric hydrolysis with formation of inositolphosphates, Ca ,-+ mobilization and protein phosphorylation. Con-A also activates the hydrolysis of phosphatidylinositol 4,5-biphosphate but bypassing the GTP-binding protein. Our results using these three different stimuli seem to indicate that, in general, the response of macrophages to produce 0 2- is decreased in aging. The decrease in the response was 48.46% with PMA, 35.5% with the chemiotactic peptide and 27.2% with Con-A. Performing a comparative study we could suggest that the process most affected in aging is the response to translocation and activation of protein kinase C to PMA. The action of N-FMLP and Con-A, activating phospholipase C and, subsequently, hydrolysis of phosphatidylinositols, is less affected, and even starts to be impaired later in the lifespan of the rat (the age-decrease using PMA was significant in 12-month-old rats while no differences between 3- and 12-month-old rats were found in macrophages stimulated with Con-A or the chemotactic peptidc). Other authors have demonstrated that macrophages from aged rats are defective in their response to other stimuli such as opsonized-zymosan and recombinant rat IFN-gamma (the production of 0 2- was 75% lower in 23-month-old rats than in 3-month-old rats) [31]. De la Fuente and Mufioz have also shown a decrease with age in the production of 0 2- in old mice peritoneal macrophage stimulated with opsonized Candida albicans [32]. There is also evidence that in old human neutrophils, cells that also exhibit respiratory burst, superoxide production is less when the cells are stimulated by latex beads [33] or a chemotactic peptide [34]. Both these stimuli act by binding to the plasma membrane and causing release of diacylglycerol and other products such as inositol trisphosphate in the initiation of the process. Indelicato et al. [35] showed that production of diacylglycerol and inositol triphosphate was impaired after FMLP stimulation of the neutrophil membrane in older patients, and a decrease in subse-

quent superoxide radical production. However, in human neutrophils Scott et al. [36] found no differences between young or old cells when stimulated with PMA, suggesting that age-related membrane changes would be related with the differences found using FMLP and latex beads and not affected by the PMA mechanism. The PMA-induced decrease in superoxide production rate in the old cells might reflect a decrease in the hexose monophosphate shunt enzymes which provide the N A D P H required for oxidase activity. The two important enzymes of this route are glucose-6-phosphatc dehydrogenase and 6-phosphogluconate dehydrogenase. We investigated if a possible decrease in the activities of these enzymes might contribute to the respiratory burst impairment found in rat macrophages with age. In fact we found a considerable, and similar, decrease of 69% and 62% in glucose-6-phosphate dchydrogenase and 6-phosphogluconate dehydrogenase, respectively. A decrease in the activities of 6-phosphogluconate dehydrogenase with age was described in rat liver and human erythrocytes [37,38]. The activation of the hexose monophosphate shunt occurs because of the increase in NADP * production during the respiratory burst. There are two sources for this NADP ÷. One is the O2--forming reaction, in which N A D P H is the electron donor. The other is the glutathione peroxidase-glutathione reductase system [39] which is responsible for the dctoxification of the H 202 that leaks back into the cytoplasm during the respiratory burst [40]. This system disposes of H202 by using it to oxidize reduced glutathione in a reaction catalyzed by glutathione peroxidase. The oxidized glutathione is then reconverted to reduced glutathione by the glutathione reductase reaction in which NADP" is produced. We investigated the effect of age on the activity of these enzymes; although no differences were found in glutathionc peroxidase activity, there was a significant decrease (approx. 45%) in glutathione reductase activity. This fact may also contribute to the decline in the respiratory burst in old animals. An age-related decrease in the activity of this enzyme have been described in the liver [41-43]. In other tissues such as lung, intestine and kidney, similar values have been described for both 3- and 24-month-old animals, or even a small increase with age [43,44]. However, in general, the behavior of glutathione pcroxidase with age is different. An increase in its levels has been found in many tissues, and has been interpreted as a compensative mechanism to counter the greater production of oxygen radicals produced with aging [44-46]. Glutathione peroxidase, catalase and SOD dismutase are the enzymes responsible tbr the removal of 0 2 and H202, to prevent the membrane and cytoplasm structures of the phagocyte itself being attacked by the oxygen-derived radicals. We therefore studied the effect of age on the catalase and C u / Z n SOD

252 dismutase activity in rat peritoneal macrophages. No variation with age was found in the C u / Z n SOD activity, however, the catalase showed a decrease of approx. 33%. The C u / Z n SOD has been described as one of the enzymes that lose activity during aging in liver and heart [47] but in brain does not change [47,48]. The catalase normally does not change with age or shows a slight increase. The negative correlation that we have found for macrophage catalase may suggest that, since the O:,- production in these cells decreases with age, the requirements of catalase might be less. In summary, the present observations show that aging produces a significant impairment of respiratory burst in rat peritoneal macrophages which may contribute to the immune system decline with age. Acknowledgements This work was supported by Grant PB89-0173 from CAI de Ciencia y Tecnologla. E.A. is a recipient of a fellowship from the Junta de Andalucia. References 1 Yoshikawa, T.T. (1983)J. Am. Geriatr. Soc. 31, 34-39. 2 Schwab. A. and Weksler, M.E. (1987) in Aging and the inmune response. (Goild, E.A.. ed.), pp. 67-81), Marcel Dekker, New York. 3 Antel, J.P., Oger, J.F., Dropcho, E., Richman, D.P., Huo, H.H. and Arnasar, B.G.W. (1980)Cell. Immunol. 54, 184-192. 4 Bender. B.S. (1985) Rev. Biol. Res. Aging 2, 143-154. 5 Dc la Fuente, M. (1985) Comp. Biochem. Physiol. 81,935-938. 6 Finger. M., Meyer, C., Winsing van Koning, H. and Emmerling. P. (1982) Gerontology 28, 223-229. 7 Babior, B.M. (1978) N. Engl. Med. 298, 659-668. 8 Babior. B.M., Kipnes, R.R. and Curnune, J.T. (1973) J. Clin. Invest. 52, 741-744. 9 Rossi. F. and Zatti, M. (1964) Experimentia 2(1, 21-23. 10 Lehmeyer, J.E., Snyderman, R. and Johnston, R.B., Jr. (1979) Blood 54, 35-45. 11 Repine, J.E.. White. J.G. and Clawson, C.C. (1974) J. Lab. Med. 83, 911-920. 12 Cohen. lt.J., Chovaniec, M.E. and Wilson, K. (1982) Bl(×)d 60, 1188-1194.

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