Metabolic and functional changes in lymphocytes and macrophages as induced by ageing

Physiology&Behavior,Vol. 53, pp. 651-656, 1993

0031-9384/93 $6.00 + .00 Copyright© 1993 PergamonPressLtd.

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Metabolic and Functional Changes in Lymphocytes and Macrophages as Induced by Ageing L. F. B. P. C O S T A R O S A , A. F. D E A L M E I D A , D. A. S A F I A N D R. C U R I l

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sdo Paulo, 05508, Butantan, Sdo Paulo, S.P., Brazil Received 26 M a r c h 1992 COSTA ROSA, L. F. B, P., A. F. DE ALMEIDA, D. A. SAFI AND R. CURl. Metabolicandfunctional changes in lymphocytes and macrophages as inducedby ageing. PHYSIOL BEHAV 53(4) 651-656, 1993.--Key enzyme activities of glycolysis,pentosephosphate pathway, Krebs cycle, and glutaminolysis were measured in lymphocytes and macrophages of 3- and 15-month-old rats from the control, thioglycollate-injected,and Walker 256 tumor-implanted groups. The percentage of phagocytosis, phagocytic index, and production of H202 in macrophages and the rates of [2-'4C]-thymidine and [5-3H]-uridine incorporation in cultured lymphocytes were also determined. The results indicate that the percentage of phagocytosis was not affected but the phagocytic index increased by twofold as a consequence of ageing, whereas the production of H202 reduced. The rates of both [2-~4C]thymidine and [5-3H]-uridine incorporation in lymphocytes from aged rats were lower as compared to those of mature animals in the three groups. Taken as a whole, the results of enzyme activities suggest that ageing may reduce the capacity for glucose utilization in lymphocytes and macrophages under the three conditions. Lymphocyte and macrophage glutamine metabolism was not markedly affected by ageing. Therefore, an impaired glucose metabolism during ageing may be one important mechanism for the alteration in lymphocyte proliferation and macrophage phagocytosis observed and also for the modification of the response to inflammatory and tumor challenges. Ageing

Lymphocyte

Macrophage

Glycolysis

Glutaminolysis

LYMPHOCYTE and macrophage metabolism has been widely investigated. Lymphocytes utilize glucose and glutamine at high rates, but these substrates are only partially oxidized: glucose is converted mainly to lactate and glutamine to glutamate, aspartate, and lactate (10,27,28), similarly to what is observed in rapidly dividing cells such as tumors (26). Although macrophages are terminally differentiated end cells in which the ability to proliferate is gradually lost (29), these cells also utilize glucose and glutamine at high rates through anaerobic glycolysis and aerobic glutamine oxidation (30). The immune response is substantially changed with ageing as reported in experimental animals and humans (4,25,32,34). The imunodeficiencies presented in aged subjects are characterized by a reduced resistance to infections (11,15) and a decreased response against tumorigenesis (12,22,35), involving both cellular and humoral responses. Reduction of secreted antibodies and diminished responses o f T and B cells to mitogens have also been described as caused by ageing. In addition, there is impaired activity of helper, cytotoxic, and suppressor T cells and lower production of cytokinins such as interleukins 2 and 3 (16,39) under this condition.

In relation to macrophages, several studies indicate that ageing provokes a delay in the response of these cells to kill tumor cells or to eliminate infectious agents in mice (14,32). The changes of macrophage phagocytosis capacity in ageing are controversial (11,40), ranging from decreased (2) to unchanged (20) and increased (31 ). Although changes in immune function are widely reported, the mechanisms involved have not been systematically examined yet. Recent reports of our laboratory have shown that ageing markedly modifies maximal activities of hexokinase, glucose-6phosphate dehydrogenase, citrate synthase, and phosphate-dependent glutaminase in several tissues, including mesenteric lymph nodes of rats (1). In addition, iymphocytes incubated and obtained from mesenteric lymph nodes of 15-month-old rats do not increase pyruvate utilization in response to Concanavalin A in opposition to 40% augment observed for cells of 3-monthold animals (36). Despite these reports, several questions remain to be answered. For instance, there are no systematic studies on key enzyme activities of glucose and glutamine metabolism in macrophages and lymphocytes during ageing. Also, whether the metabolic and functional responses of these cells to certain chal-

' Requests for reprints should be addressed to R. Cuff.

651

652

COSqA ROSA ET AL.

lenges such as thioglycollate injection and tumor implantation in the peritoneal cavity are modified due to the ageing process has not been examined yet. The effect of ageing on lymphocyte and macrophage responses to inflammatory and tumor challenges was examined in vivo. For this purpose. 1. macrophage phagocytic capacity and production of H202, 2. rates of proliferation of T and B lymphocytes, and 3. metabolism of glucose and glutamine by measuring key enzyme activities of their metabolic pathways were evaluated. Activities of hexokinase--which provides a quantitative index of maximum glycolytic flux, of glucose-6phosphate dehydrogenase--which provides a quantitative index of the flux through the pentose-phosphate pathway, of phosphatedependent glutaminase--which permits obtaining a quantitative index of the maximum flux through glutaminolysis, and of citrate synthase--which provides a qualitative index of the flux through the Krebs cycle, were measured in peritoneal macrophages and mesenteric lymphocytes obtained from adult and aged rats injected with thioglycollate or implanted with Walker 256 tumor cells in the intraperitoneal cavity. The rates of incorporation of [2-~4C]-thymidine and [5-SH]-uridine into lymphocytes cultured in the presence of concanavalin A (T cells) and lipopolysaccharide (B cells) were determined as indicative of the capacity of B and T lymphocyte proliferation under these conditions. METHOD

Animals Male albino rats 3 (mature) and 15 (aged) months old were obtained from Butantan Institute. The rats were kept in an environment at 23°C and a light/dark cycle of 12/12 h; lights on from 0700 h.

Chemicals and Enzyme~ All chemicals and enzymes were obtained from Boehringer Mannheim, Germany, except for the followings: [2-~4C]-thy midine and [5-3H]-uridine, which were purchased from Amersham, UK, concanavalin A (Con A) and lipopolysaccharide (LPS) and phorbol-myristate-acetate (PMA) from Sigma (USA).

Treatments Thioglycollate-treated rats (TG) were intraperitoneally injected with 3 ml of a 4% solution of thioglycollate in saline, 4 days before the harvest procedure. Tumor-bearing rats (TB) received 1 ml of a suspension of 105 Walker 256 tumor cells in 0.9% saline, 7 days before the experiment. Tumor cells were obtained from solid tumor after 2 weeks of a subcutaneous injection of 2 × 10v cells on the left flank of the rats. All the results were compared with control animals (C), which were not submitted to any treatment.

Experimental Procedure After 4 days of thioglycollate injection and 7 days of intraperitoneal implantation of tumor cells, the rats were killed by decapitation without anesthesia. Maerophages of the intraperitoneal cavity were removed (5) and separated from ascite tumor cells through a Ficoll-Hypaque gradient. Mesenteric lymph nodes were dissected and lymphocytes isolated as described by Vieira et al. (38).

MacrophaL, e Pha~,,oql,to.vis Macrophages were incubated with l0 ml of PBS (phosphatebuffer saline) containing opsonized zymosan for 30 min at 37°C. The percentage of phagocytosis was determined by counting (in a counting chamber) the cells that had phagocytosed the particles. A phagocytosis index was also calculated as follow: (~!/,of cells presenting phagocytosis) × (number of phagocytosed particles)/ 1000.

Production oI' H:O~ The production of hydrogen peroxide was monitored by horseradish peroxidase (HRPO)-dependent oxidation of phenol red as described by Pick and Mizel (33). The values are presented as nmol/106 cells.

Incorporation o1[2-J4(]_ Thymidine and [5-SH]- Uridine Into Cultttred L ymphoc3,tes Cervical lymphocytes were cultured in RPMI- 1640 medium for 24 h at 37°C in an artificially humidified atmosphere of 5% CO: in air at sterile conditions. Cultures were performed in a LAB-LINE Microprocessor CO, incubator (LAB-LINE, USA) in 96-welt plates (Corning, NY, USA), 1 × 10~ cells per well (total volume, 200 ul). After 24 h in culture, more than 98% of lymphocytes were viable, as measured by exclusion of trypan blue dye. Con A- and LPS-activated lymphocytes were obtained by the addition of 20 ul of the mitogens in alcoholic solution per well at final concentrations of 5 ug/ml and 100 ug/ml, respectively. These concentrations provide maximal responses under the conditions described herein (23,24). Quiescent cell groups (no addition) received an equal volume of ethanol in PBS (final concentration of 0.05%). After 24 h, cells were pulsed with 20 ul of 0.02 uCi [ 2 - t 4 C ] thymidine (sp. act. 56.0 mCi/mmol) and 0.3 #Ci [5-3H]-uridine (sp. act. 26.0 Ci/mmol) diluted in sterile PBS having both a final concentration of I ug/ml. Cells were then maintained under this condition for additional 15 h and automatically harvested by using a multiple cell harvester and filter papers cat. No. 11731 (Skatron Combi, Suffolk, UK). The paper discs containing labelled cells were taken to be counted in 5 ml of Bray's scintillation cocktail, consisting of 60 g/l naphthalene, 4 g/l 2,5-diphenyloxazole (PPO), 200 mg/I 1,4-di-[2-(5-phenyloxazolyl)]-benzene (POPOP), 10~ methanol (by vol.), and 2% ethylene glycol (by vol.) in p-dioxan (chromatographic grade) in a Beckman-LS 5000TD liquid scintillator (Beckman Instruments, Fullerton, CA, USA). All reagents used in the preparation of Bray's were from Sigma (USA) or Merck (Darmstadt, Germany).

Enzyme Assays Enzyme activities were determined as previously described (1,9,29,37). The extraction medium for hexokinase (EC 2.7.1.1 ) contained 25 mM Tris-HC1, 1 m M EDTA, and 30 mM mercaptoethanol at pH 7.4, and that for glutaminase (EC 3.5.1.2) contained 150 m M potassium phosphate, l m M EDTA, and 50 mM Tris-HCl at pH 8.6. The extraction medium for citrate synthase (EC 4.1.3.7) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) contained 50 m M Tris-HC1 and 1 m34 EDTA, the final pH was 7.4 and 8.0, respectively. For all enzymes, 0.05% (v/v) Triton X-100 was added to the assay system to complete the extraction of the enzymes. The final volume of the assay mixtures in all cases was 1.0 ml. Citrate synthase was assayed by following the rate of change in A-412, whereas the remainder of the enzymes was assayed at A-340. All measurements were

AGEING EFFECTS ON LYMPHOCYTE AND MACROPHAGE METABOLISM

performed in a Gilford (Response) recording spectrophotometer at 25 °C, except for glutaminase, which was determined at 37 °C. For all enzymes studied, preliminary experiments established that extraction and assay procedures were such as to produce maximum enzyme activities (7). The enzyme activities are expressed as nmol of substrate utilized/min per 101° cells.

Statistical Analysis The results are presented as mean and standard error of the mean (SEM). The paired t-test was used and differences were considered significant at p < 0.05. RESULTS AND DISCUSSION The percentage of phagocytosis was not modified as a consequence of ageing in the three groups studied: control (C), thioglycollate-injection(TG), and implantation of Walker 256 tumor cells (TB). However, the phagocytic index was always higher in macrophages of aged for the C (2.3-fold), TG (67%), and TB (42%) groups. Findings obtained by others (14) also indicate that macrophages from aged rats present higher capacity to phagocytose several particles in the same cell. The significance of this fact for macrophage function remains unknown. Thioglycollate injection enhanced phagocytic index in mature (3.2-fold) and aged (62%) rats, whereas the percentage of phagocytosis augmented by 1.5-fold in both, as compared to the control group. Similar response was found by implantation of Walker 256 tumor cells. Therefore, ageing does not affect phagocytosis capacity when macrophages are stimulated by thioglycollate injection or tumor cell implantation. In opposition, the production of H202 by tumor-activated macrophages was markedly reduced in aged rats either in the absence (32%) and in the presence (35%) of PMA. The decreased activity of glucose-6-phosphate dehydrogenase in macrophage from aged rats (Table 3) may be involved in these observations (3,19,21). Indeed, this enzyme produces NADPH, which is a precursor for H202 formation. Despite this, however, the role of other mechanisms have to be investigated, such as the production of H202 by PMA stimulus via protein kinase C and NADPH oxidase activity (6). Addition of Con A to the culture medium, as compared to no addition, elevated the rates of [2Y4C]-thymidine and [5-3H]uridine incorporation into lymphocytes from mature and aged rats of the C, TG, and TB groups (Table 2). Similar findings were obtained for the C and TG groups in the presence of LPS, except for the TB group, which showed a noticeable decrease of thymidine and uridine incorporation in lymphocytes from mature and aged rats. These observations indicate that the response o f T lymphocytes to Con A is not altered by ageing even in cells obtained from the TG and TB groups. However, the rates of thymidine and uridine incorporation surprisingly reduced in B lymphocytes (LPS) of the group implanted with tumor cells (TB). The significance of this fact remains to be elucidated. The rates of incorporation of [2-~4C]-thymidine and [5-3H]uridine into lymphocytes from aged rats diminished, as compared to those of mature animals, under control condition (no addition) and in the presence of Con A and LPS (Table 2). So far, B and T lymphocytes (as indicated by LPS and Con A stimuli, respectively) from aged rats presented lower capacity to proliferate, as compared to those from mature animals. This is an interesting observation and supports the idea that the function of both B and T lymphocytes is impaired by ageing process (15,35). Thioglycollate injection and implantation of Walker 256 tumor cells raised the rates of [2-'4C]-thymidine and [5-3H]-uridine

653

incorporation into lymphocytes from mature and aged rats cultured in the absence and in the presence of the mitogens with few exceptions (Table 2); incorporation of both nucleotides into T cells of mature rats of the TG group and ofthymidine into B cells of aged rats of the TB group. Taken together, the two challenges enhanced the rates of proliferation of B and T lymphocytes, and this effect was not markedly modified by ageing. Whether the changes above in macrophage and lymphocyte function were accompanied by important modifications in glucose and glutamine metabolism was then examined. Hexokinase is assumed to control the flux of substrates through glycolysis ( 1,17,18,29). Ageing process reduced this enzyme activity in lymphocytes from the C and TG groups and increased by twofold in TB rats, whereas in macrophages there was a decrease in the TG and TB groups. These findings suggest lower capacity for glucose utilization in both cell types as a consequence of ageing. It was notable the fact that thioglycollate injection and implantation of tumor cells raised hexokinase activity by twofold in macrophages. In opposition, this enzyme activity was lowered in lymphocytes from mature rats of the TG (22%) and TB (61%), as compared to the C group. These observations suggest an enhanced capacity for glucose utilization in macrophages and reduced in lymphocytes due to thioglycollate injection and tumor cell implantation. The degree of this response seemed to be dependent on the age of the animals. The activity of the pentose-phosphate pathway is regarded to be regulated by glucose-6-phosphate dehydrogenase activity ( 1,17,18,29). The ageing process diminished this enzyme activity in lymphocytes of the three groups and in macrophages of TG and TB rats. This fact might lead to impaired function of both cells, especially in terms of H202 production as previously mentioned (Table 1). Thioglycollate injection caused a reduction of glucose-6-phosphate dehydrogenase activity in lymphocytes from aged rats, whereas implantation of tumor cells diminished in both mature and aged rats. The mechanisms to explain these changes remain unknown. Macrophage glucose-6-phosphate

TABLE 1 PERCENTAGE OF PHAGOCYTOS1S,PHAGOCYTICINDEX, AND PRODUCTION OF HYDROGEN PEROXIDES BY MACROPHAGES Walker

Control Percentage of phagocytosis Mature 39 ___2.0 Aged 40 _+ 1.0 Phagocytic index Mature 1.20 _+0.02 Aged 2,71 _+0.03t Control

Thioglycollate

60 _+ 1.0" 58 _+ 1.0"

58 + 2.0* 56 _ 3.0*

2.63 + 0.05 4.40 + 0.01 t

2.81 ___0.08 3.98 + 0.10

Thioglycollate

Production of hydrogen peroxides Mature --Aged ---

Tumor

No Addition

PlusPMA

22.4 _+0.7 15.3 _+0.9§

51.0 + 0.6~ 33.1 + 0.7t§

Macrophages were obtained from the intraperitoneal cavity of the three groups. For details see Method section. The values are presented as mean + SEM. PMA: phorbol-myristate-acetate. * p < 0.05 compared to the control group. t P < 0.05 compared to mature rats of the same group. p < 0.05 different due to the effect of PMA on hydrogen peroxide production in macrophages from the TB group.

654

COSTA ROSA ET AI

TABLE 2 RATES OF [2-M(]-THYMIDINE AND [5-SH]-URID1NE INCORPORATION IN CULTURED I.YMPHOCYTES No Addition

Plus ('on. A

[2-~4C]-Thymidine incorporation (DPM) Mature C 2009.8 ± 20.2 6215,6 TG 3743.8 ± 33.1+ 6330,9 TB 4921.9 _+ 33.3t 7424.2 Aged C 1402.2 ± 41.35 3288.6 TG 2851,3 _+ 33.7t~ 4664.4 TB 3997,4 ± 52.6t5 5054.6 [5-3H]-Uridine incorporation (DPM) Mature C 714,3 _+ 27.4 1936.1 TG 1251.8 ± 19.0+ 1941.7 TB 1665.1 ± 14.7+ 2379.5 Aged C 476.9 ± 8.45 1137.7 TG 1000.4 _+ 18.8t5 1485.4 TB 1337.3 ± 14.6t5 1774.4

Plus LPS

± 36.7* _+ 41.2" ± 46.3* t

4407.3 +_ 33.3* 5218.8 _+ 46.7" t 4605.0 +_ 50.0* t

± 25.7*5 + 52.0*+5 ± 31.9"+ 5

2626.2 ± 46.7* 5 3220.2 ± 60.2*+ 5 2698.7 _+ 42.4* 5

± 17.1" + 17.9' +_ 2 1 . 8 " t

1304.8 ± 24.6* 1762.8 ± 15.4"+ 1545.0 ± 14.4"t

± 22.6*5 _+ 13.2"75 ± 15.0"t5

849.8 _+ 21.9"$ 1684.2 +_ 17.5"I 936.8 +_ 25.6"?5

For details of the procedure see Method section. The values are presented as mean ± SEM of 12 wells and two separated experiments. * p < 0.05 due to addition of Con A and LPS, as compared to control condition (no addition). t p < 0.05 compared to the control group. 5 P < 0.05 for the comparison between mature and aged rats of the same group.

TABLE 3 MAXIMAL ACTIVITIES (nmol/min PER 10~° CELLS) Lymphocytes Mature

Macrophages Aged

Hexokinase C 65.7 ± 5.1 31.9._+ 0.7* TG 50.7 _+ 1.1 -[ 28.8 ± 0.9* TB 25.4 ± 1.07 54.4 ± 1.7'+ Glucose-6-phosphatase dehydrogenase C 83.2 +_ 6.1 76.8 _+ 3.9 TG 84.1 +_ 1.2 49.5 ± 1.6"~ TB 64.2 + 1.3t 45.2 ± 13.*+ Citrate synthase C 298.3 ± 10 211.8 ± 9.2. TG 181.4 + 1.3t 141.1 -+ 1.4*t TB 162.7 ± 1.4~ 102.7 + 1.8"+ Phosphate-dependent glutaminase C 45.9 _+ 2.9 48.7 ± 1.5 TG 65.1 ± 3.2t 81.6 +_ 3 . 4 " t TB 43.8 ± 1.8 66.8 ± 3 . 8 " t

Mature

287.3 ± 4.3 632.2 ± 13t 562.3 _+ 3.2+

Aged

278.0 ± 2.3 499.4 ± 5 . 9 " t 461.1 + 2 . 4 " t

8.2 +_ 0.8 14.4 +_ 1.0+ 19.4 ± 1.0#

8.0 _+ 1.0 8.6 ± 0.4t t0.6 ± 0.7+

36.0 ± 1.0 54.5 _+ 4.2? 56.2 ± 4.1"

32.7 + 1.1 36.2 ± 1.3"? 16.3 ± 1.5*t

144.3 ± 2.7 98.0 + 2.7¢ 183.7 _+ 1.1"

99.2 _+ 1.3" 130.4 _+ 3 . 6 " t 67.8 +_ 1.2*t

C, control group. TG, thioglycolate-injected group. TB, Walker 250 tumor cell implanted group. For details of the enzyme assays see Method section. The values are presented as mean +_ SEM of eight determinations from two to three experiments. * p < 0.05 compared to mature rats. t P < 0.05 different due to thioglycollate injection or tumor cell implantation as compared to the control group.

AGEING EFFECTS ON LYMPHOCYTE AND MACROPHAGE METABOLISM

dehydrogenase activity raised by thioglycollate injection and tumor cell implantation in mature rats and did not alter in aged animals. Therefore, the metabolic response of macrophages to both challenges was abolished as a consequence of ageing. Citrate synthase activity provides qualitative information on the flux of substrates through the Krebs cycle (1,17,18,29). This enzyme presented lower activity in iymphocytes and macrophages of 15-month-old, as compared to mature rats, from the three groups. This fact is evidence that a low capacity for the flux of substrates through the Krebs cycle may occur in both cells due to ageing. The stimuli of thioglycollate and tumor cells diminished this enzyme activity in lymphocytes of mature and aged rats and caused an increment in macrophages of mature animals. In aged rats, however, thioglycollate showed a tendency to raise (11%) citrate synthase activity, whereas in the tumor implanted group there was a significant decrease (50%). The flux of substrates through glutaminolysis in lymphocytes and macrophages is controlled by phosphate-dependent glutaminase activity (10,27,29). Due to ageing, this enzyme activity increased in lymphocytes of the TG (25%) and TB (53%) groups, whereas in macrophages there was an increase in the TG (33%) and a decrease in the C (46%) and TB (63%) groups. Despite being important for cell division (27,28), the reduced proliferation of B and T lymphocytes during ageing (Table 2), is hardly due to a low capacity for glutamine utilization by these cells. However, an impairment of glutamine metabolism could well be one important mechanism for the altered macrophage function reported by others (2,14) in aged rats.

655

Thioglycollate injection enhanced glutaminase activity in lymphocytes of mature (42%) and aged (68%) rats and in macrophages of 15-month-old animals (32%), whereas this stimulus diminished this enzyme activity in macrophages from mature rats (47%). Implantation of Walker 256 tumor cells did not affect glutaminase activity in lymphocytes from 3-month-old rats but elevated by 37% in aged animals. In macrophages, there were a trend to raise (27%) in mature rats and to decrease (32%) in aged animals. These results indicate that the response of glutaminase activity to thioglycollate and tumor stimuli markedly varied with the age of the animals. Taken as a whole, ageing process lowered the activities of hexokinase, glucose-6-phosphate dehydrogenase and citrate synthase in lymphocytes and macrophages and did not consistently modify that of glutaminase. These findings suggest a depressed capacity of lymphocytes and macrophages for glucose utilization in aged rats and may be one important mechanism for the alterations in macrophage phagocytic index and B and T lymphocyte proliferation observed. Furthermore, however, ageing modified the metabolic and functional responses of these cells to inflammatory and tumor challenges. Whether these changes were due to hormonal factors (8,13) remains to be investigated. ACKNOWLEDGEMENTS The authors are grateful to the technicalassistanceofJ, R. Mendonca and G. de Souza. This research is supported by CNPq, FAPESP, and Sandoz Foundation for Gerontological Research.

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